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US20210093654A1 - Pharmaceutical composition for preventing or treating liver cancer - Google Patents

Pharmaceutical composition for preventing or treating liver cancer Download PDF

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US20210093654A1
US20210093654A1 US16/634,675 US201816634675A US2021093654A1 US 20210093654 A1 US20210093654 A1 US 20210093654A1 US 201816634675 A US201816634675 A US 201816634675A US 2021093654 A1 US2021093654 A1 US 2021093654A1
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dsrna
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porous silica
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Cheolhee WON
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Lemonex Inc
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating liver cancer.
  • HCC Hepatocellular carcinoma
  • HCC human Crohn's disease
  • HCC is also characterized by multiple tumor progression.
  • the damaged liver tissues in the early stage evolve to small nodular hypercellular lesions called dysplastic nodules (DNs).
  • DNs small nodular hypercellular lesions
  • eHCC early hepatocellular carcinoma
  • eHCC early hepatocellular carcinoma
  • the lesion would be subjected to treatment if it is determined as a cancer by histological diagnosis.
  • eHCC generally exhibits minimal dysplasia and lacks clear invasive or destructive growth. Therefore, even for hepatopathologists, it is often difficult to distinguish recurrent nodules, precancerous lesions and early lesions. Due to such reasons, discovery of objective molecular markers that standardize histological diagnosis of eHCC and induce appropriate therapy is eagerly required.
  • the discovery of biomarkers related to accurate HCC diagnosis may facilitate identification of precancerous lesions possibly progressing to HCC and to determine surgically resectable lesions, thereby supporting a surgeon to design a surgical range in HCC patients. Identifying additional molecular markers that predict possible occurrence of HCC in precancerous lesions may be helpful for identifying patients at risk for recurrence following surgical resection.
  • the present study is intended to establish a gene selection strategy to identify potential causative genes by combining clinicopathological and gene expression data of staged hepatocarcinoma tissues defined by hepatopathologists. As a result, 10 genes expected to be the cause of early stage HCC could be selected.
  • HCC diagnostic markers that is, BANF1, PLOD3 and SF3B contribute to early malignant transformation of hepatocytes in formation of hepatic tumor and are also targets for molecular therapy of liver malignancy.
  • a pharmaceutical composition for preventing or treating liver cancer including: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 12, 14 to 19, 21, 23, 24, 26, 28 to 34, 35 to 37, 39 to 41, 43, 45 to 47, 49 to 53, 55 to 60, 62 to 73, 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 165, 167 to 172, 174, 176, 177, 179, 181 to 187, 188 to 190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to 226, 228 to 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28, and an antisense
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310.
  • siRNA or dsRNA is loaded on at least one carrier selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin, lipid particles, polyethylene glycol, poly-L-guluronic alginate, polyglycolic-polylactic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and hydrogel.
  • carrier selected from the group consisting
  • the carrier is a porous silica particle characterized in that t when a ratio of absorbance in the following Equation 1 becomes 1 ⁇ 2 is 20 or more:
  • a 0 absorbance of the porous silica particle measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particle into a cylindrical dialysis membrane having pores with a diameter of 50 kDa
  • a t is absorbance of the porous silica particle measured after t hours elapses from the measurement of A 0 ).
  • composition according to the above 8 wherein the t is 40 or more.
  • siRNA includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 28, 119 and 136, and an antisense RNA having a complementary sequence thereto, and
  • the dsRNA has at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 181, 272 and 289.
  • porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
  • porous silica particle has at least one hydrophilic substituent selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate, polyethylene glycol, substituted or unsubstituted C 1 to C 30 alkyl, substituted or unsubstituted C 3 to C 30 cycloalkyl, substituted or unsubstituted C 6 to C 30 aryl, and C 1 to C 30 ester groups.
  • hydrophilic substituent selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl
  • porous silica particle is positively or negatively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
  • porous silica particle is positively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
  • porous silica particle has an average particle diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
  • the pharmaceutical composition of the present invention provides preventive and therapeutic effects of liver cancer by specifically knocking down the genes expressed in early stage liver cancer cells, so as to prevent the development of liver cancer and inhibit the metastasis and proliferation of liver cancer cells.
  • FIG. 1 is views illustrating results of measuring expression levels of indicator factors (“markers”) corresponding to siRNAs by Western blotting, respectively, after in vitro transfection of siRNAs to Hepa-1c1c7 and SNU-449 cell lines in EXAMPLE 1 by methods described in EXAMPLE 1-2 or 1-8, wherein each siRNA includes a sense RNA having a sequence in Table 11 and an antisense RNA having a complementary sequence to that of the sense RNA.
  • FIG. 2 is views illustrating results of analyzing migration and invasion of markers corresponding to siRNAs by a method described in EXAMPLE 1-5 and analyzing scratch wound healing ability of the same by a method described in EXAMPLE 1-6, respectively, after in vitro transfection of siRNAs to SNU-449 cell line in EXAMPLE 1-1 by a method described in EXAMPLE 1-2, wherein each siRNA includes a sense RNA having a sequence in Table 12 and an antisense RNA having a complementary sequence to the sequence of the sense RNA.
  • a of FIG. 3 is views illustrating results of analyzing expression levels of markers corresponding to siRNAs and EMT regulatory proteins by a method described in EXAMPLE 1-9, respectively, after in vitro transfection of siRNAs to SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2, wherein each siRNA includes a sense RNA having a sequence in Table 12 and an antisense RNA having a complementary sequence to the sequence of the sense RNA; and B of FIG. 3 is views illustrating the analyzed results of hepatic tumor sizes and survival rates of mice, after subcutaneous injection of transfected cells in the above (A) into athymic nude mice.
  • a of FIG. 4 is views illustrating processes of in vivo transfection of siRNAs by a method described in EXAMPLE 1-8, ultrasonic images and the number of tumors over time, wherein each siRNA includes a sense RNA having a sequence in Table 13 and an antisense RNA having a complementary sequence to the sequence of the sense RNA; and B of FIG. 4 is a view illustrating results of analyzing expression inhibitory levels of siRNAs loaded on porous nanoparticles to indicator genes corresponding to the siRNAs by the method described in EXAMPLE 1-9.
  • FIG. 5 is micrographs illustrating porous silica particles.
  • FIG. 6 is micrographs illustrating porous silica particles.
  • FIG. 7 is micrographs illustrating small pore particles during production of the porous silica particles.
  • FIG. 8 is micrographs illustrating small pore particles.
  • FIG. 9 is micrographs demonstrating biodegradability of porous silica particles.
  • FIG. 10 is a view illustrating a tube having a cylindrical dialysis membrane.
  • FIG. 11 is a graph illustrating results of absorbance reduction of the porous silica particles over time.
  • FIG. 12 is a graph and a table illustrating results of absorbance reduction by particle diameter of the porous silica particles over time.
  • FIG. 13 is a graph and a table illustrating results of absorbance reduction by pore diameter of the porous silica particles over time.
  • FIG. 14 is a graph illustrating results of absorbance reduction of the porous silica particles by pH in environments over time.
  • FIG. 15 is a graph illustrating results of absorbance reduction of the porous silica particles over time.
  • FIG. 16 is a view illustrating a tube for identifying release of bioactive material from the porous silica particles.
  • FIG. 17 is a graph illustrating release of bioactive material loaded on the porous silica particles over time.
  • FIG. 18 is micrographs demonstrating siRNA release in mice by loading the porous silica particles with siRNA.
  • siRNA refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing.
  • siRNA can inhibit expression of a target gene and may be provided as an efficient gene knockdown method or as a gene therapy method.
  • the siRNA molecule may have a structure in which a sense strand (a sequence corresponding to mRNA sequence of the target gene) and an antisense strand (a complementary sequence to mRNA sequence of the target gene) are located on sides opposite to each other to form a double chain.
  • siRNA molecules may have a single stranded structure with self-complementary sense and antisense strands.
  • the siRNA is not limited to a complete pair of double-stranded RNA modalities that are paired with each other, but may also include modalities that are not paired due to a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) or the like.
  • the siRNA terminal structure may include blunt or cohesive terminals as long as it can inhibit expression of a target gene by RNA interference (RNAi) effects.
  • the cohesive terminal structure may be a 3′-terminal protruding structure and 5′-terminal protruding structure.
  • siRNA molecules may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and the antisense strands.
  • the siRNA molecule formed by expression of the nucleotide sequence may form a hairpin structure by intramolecular hybridization, which in turn forms a stem-and-loop structure on the whole. This stem-and-loop structure may be processed in vitro or in vivo to produce siRNA molecules capable of mediating RNAi.
  • dsRNA refers to a siRNA precursor molecule that meets a RISC complex containing DICER enzyme (Ribonuclease III) of a target cell and is cleaved into siRNA. In this process, RNAi is generated. dsRNA has a longer sequence by several nucleotides than siRNA and may have a structure wherein a sense strand (a sequence corresponding to mRNA sequence of the target gene) and an antisense strand (a sequence complementary to mRNA sequence of the target gene) are located on sides opposite to each other to form a double chain.
  • Nucleic acid may include any DNA or RNA, for example, chromosomes, mitochondria, viruses and/or bacterial nucleic acids present in a tissue sample. One or both strands of a double-stranded nucleic acid molecule may be included, and further any fragment or portion of an intact nucleic acid molecule.
  • Gene refers to any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expressions.
  • the gene may include only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein.
  • the nucleic acid sequence may involve gene abnormality in exon, intron, an initiation or termination region, a promoter sequence, another regulatory sequence or a specific sequence adjacent to the gene.
  • the term “gene expression” generally refers to a cellular process in which a polypeptide having biological activity is produced from a DNA sequence and exhibits biological activity in the cell.
  • the gene expression may include not only transcription and translation processes but also post-transcription and post-translation processes that may affect the biological activity of the gene or gene product. Such processes may include polypeptide synthesis, transport and post-translational modification as well as RNA synthesis, processing and transport, but it is not limited thereto.
  • the term “gene expression” refers to a process in which a precursor siRNA is produced from a gene.
  • siRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on a protein coding gene. Nevertheless, the formation of mature siRNAs from siRNA genes may be encompassed by the term “gene expression” as that term is used herein.
  • target gene refers to a gene targeted for modulation using the method and composition in the subject matters disclosed herein. Therefore, the target gene includes a nucleic acid sequence with a specific expression level down-regulated by siRNA into mRNA or a polypeptide level.
  • target RNA or “target mRNA” refers to a transcript of the target gene that is bound to siRNA and induces modulation of expression in the target gene.
  • transcription refers to a cellular process involving interaction between an expression-inducible gene, which is RNA of structural information present in a coding sequence of the gene, and RNA polymerase.
  • down-regulation refers to considerably decreasing the expression of a specific gene into mRNA or the expression level into a protein by gene transcription or gene translation in activated cells, as compared to normal tissue cells.
  • the term “treatment” means an approach to obtain beneficial or desired clinical results.
  • the beneficial or desired clinical results may include, without limitation, alleviation of symptoms, reduction in an extent of disease, stabilization (i.e., not worsening) of disease state, delayed progression of disease or reduction in progress rate of disease, improvement, temporary mitigation and alleviation of disease state (partially or wholly), whether or not it is detectable.
  • the term “treatment” may also refer to increasing the survival rate compared to that expected survival when untreated.
  • the treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments may include treatments required for disorders that have already occurred as well as disorders to be prevented.
  • prevention means any action to inhibit or delay development of a related disease. It will be apparent to those skilled in the art that the composition mentioned herein may prevent initial symptoms, or related diseases in a case of administering before symptoms appear.
  • the present invention provides a pharmaceutical composition for preventing or treating liver cancer, that is, hepatocellular carcinoma (HCC), which includes: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, as well as antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.
  • the siRNA or dsRNA of the present invention may be derived from animals including humans, such as monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, and the like, and is preferably derived from humans.
  • siRNA or dsRNA of the present invention may be modified by deletion, substitution or insertion of a functional equivalent of nucleic acid molecule constituting the siRNA or dsRNA, for example, a part of the base sequence in the siRNA or dsRNA of the present invention, however, may also be a concept including variants which are capable of functionally performing the same action as the siRNA or dsRNA of the present invention.
  • the siRNA or dsRNA of the present invention may be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or may include commercially available ones. Further, the composition of the present invention may include not only siRNA or dsRNA itself of the present invention but also other substances, for example, compounds, natural products, novel proteins, etc. which are capable of increasing an expression rate of the siRNA or dsRNA of the present invention in cells.
  • siRNA or dsRNA of the present invention may be provided in a state of being included in a vector for intracellular expression.
  • the siRNA or dsRNA of the present invention may be introduced into cells by various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and a nuclear protein, a complex of DNA and lipid and the like.
  • the siRNA or dsRNA of the present invention may be provided in a form of being contained in a carrier enabling efficient introduction into a cell.
  • the carrier is preferably a vector, and both viral vectors and non-viral vectors are usable.
  • the viral vector may include lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector, etc., preferably, is a lentivirus vector, but it is not limited thereto.
  • Lentivirus is one type of retrovirus characterized by infecting a non-mitotic cell as well as a mitotic cell due to nucleophilic property of pre-integrated complex (a virus “shell”) that allows active incorporation into nucleopores or a complete nuclear membrane.
  • a virus “shell” pre-integrated complex
  • the vector containing siRNA or dsRNA of the present invention preferably includes a selection marker.
  • the “selection marker” is intended to facilitate selection or screening of cells into which siRNA or dsRNA of the present invention has been introduced.
  • the selection marker used in the vector is not particularly limited as long as it is a gene capable of easily detecting or determining whether or not the vector was introduced.
  • examples thereof may typically include markers endowing selectable phenotypes such as drug resistance, auxotrophy, tolerance to cytotoxic agents, expression of surface protein, etc., in particular, green fluorescent protein (GFP), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hisD), guanine phosphoribosyltransferase (Gpt) or the like.
  • GFP green fluorescent protein
  • puromycin puromycin
  • Neo neomycin
  • Hyg hygromycin
  • hisD histidinol dehydrogenase gene
  • Gpt guanine phosphoribosyltransferase
  • Gpt guanine phosphoribosyltransferase
  • composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28 in Table 1 below, and an antisense RNA having a complementary sequence thereto; and dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181 in Table 1 below.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28 in Table 1 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181 in Table 1 below, may target variant 1 sequence (SEQ ID NO: 1) of human BANF1 gene to inhibit expression of the human BANF1 gene variant 1 through RNAi, thereby achieving effects of preventing or treating HCC.
  • Target sequence 5 GC content: 45.0% 5′-CAA GAA GCT GGA SEQ ID NO: 5 GGA AAG-3′ (SEQ ID Sense strand: NO: 482) 5′-CAA GAA GCU GGA GGA AAG (Position in UU-3′ gene sequence: 601) SEQ ID NO: 326 Antisense strand: 5′- CUU UCC UCC AGC UUC UUG UU-3′ SEQ ID NO: 158 dsRNA: 5′- CAA GAA GCU GGA GGA AAG UU UCU AAA G-3′
  • Target sequence 6 GC content: 40.9% 5′-GAA AGA TGA AGA SEQ ID NO: 6 CCT CTT CC-3′ (SEQ Sense strand: ID NO: 483) 5′-GAA AGA UGA AGA CCU CUU (Position in CCU U-3′ gene sequence: 667) SEQ ID NO: 327 Antisense strand: 5′-GGA AGA GGU CUU
  • composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55 in Table 2 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208 in Table 2 below.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55 in Table 2 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208 in Table 2 below, may target variant 2 sequence (SEQ ID NO: 2) of human BANF1 gene to inhibit expression of the human BANF1 gene variant 2 through RNAi, thereby achieving effects of preventing or treating HCC.
  • Target sequence 29 GC content: 40.9% 5′-ATG AC A ACC TCC SEQ ID NO: 29 CAA AAG CA-3′ (SEQ Sense strand: ID NO: 506) 5′-AUGACAACCUCCCAAAAGCA (Position in UU-3′ gene sequence: 452) SEQ ID NO: 350 Antisense strand: 5′-UGCUUUUGGGAGGUUGUCAU UU-3′ SEQ ID NO: 182 dsRNA: 5′-AUGACAACCUCCCAAAAGCA UU UCU AAA G-3′ Target sequence 30: GC content: 40.9% 5′-CCG AGA CTT CGT SEQ ID NO: 30 GGC AGA-3′ (SEQ ID Sense strand: NO: 507) 5′- CCGAGACUUCGUGGCAGA (Position in UU-3′ gene sequence: 472) SEQ ID NO: 351 Antisense strand: 5′-UCUGCCACGAAGUCUCGG UU-3′ SEQ ID NO: 183 d
  • Antisense strand 5′-UUAGGCUUGACGGACCUGG UU-3′
  • SEQ ID NO: 201 dsRNA: 5′- CCAGGUCCGUCAAGCCUAA UU UCU AAA G-3′
  • Target sequence 49 GC content: 45.0% 5′-GCA GCA GAG AAA SEQ ID NO 49 GGA AGT-3′ (SEQ ID Sense strand: NO: 526) 5′- GCAGCAGAGAAAGGAAGU (Position in UU-3′ gene sequence: 182) SEQ ID NO: 370
  • Antisense strand 5′-UACUUCCUUUCUCUGCUGC UU-3′
  • SEQ ID NO: 202 dsRNA: 5′- GCAGCAGAGAAAGGAAGU UU UCU AAA G-3′
  • Target sequence 50 GC content: 45.0% 5′-CCT ATC TCC CTC SEQ ID NO: 50 AGA ACT-3′ (SEQ ID Sense strand: NO: 527) 5′- CCUAUCUCCC
  • composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120 in Table 3 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273 in Table 3 below.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120 in Table 3 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273 in Table 3 below, may target a sequence of human PLOD3 gene (SEQ ID NO: 3) to inhibit expression of the human PLOD3 gene through RNAi, thereby achieving effects of preventing or treating HCC.
  • SEQ ID NO: 3 human PLOD3 gene
  • Target sequence 56 5′- GC content: 50.0% CCA GAG AAG CTG SEQ ID NO: 56 Sense strand: 5′- CTG GTG AT-3′ (SEQ CCAGAGAAGCUGCUGGUGAU UU-3′ ID NO: 533) (Position in SEQ ID NO: 377 Antisense strand: 5′- gene sequence: 562) AUCACCAGCAGCUUCUCUGG UU-3′ SEQ ID NO: 209 dsRNA: 5′-CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3′ Target sequence 57: 5′- GC content: 55.0% CCA CAG CTG AAA SEQ ID NO: 57 Sense strand: 5′-CCACAGCUGAAACCGAGG CCG AGG-3′ (SEQ ID UU-3′ NO: 534) (Position in SEQ ID NO: 378 Antisense strand: 5′- gene sequence: 590) CCUCGGUUUCAGCUGUGG UU-3′ SEQ ID NO:
  • composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157 in Table 4 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310 in Table 4 below.
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157 in Table 4 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310 in Table 4 below, may target a sequence of human SF3B4 gene (SEQ ID NO: 4) to inhibit expression of the human SF3B4 gene through RNAi, thereby achieving effects of preventing or treating HCC.
  • SEQ ID NO: 4 sequence of human SF3B4 gene
  • Target sequence 121 5′- GC content: 40.9% AAT CAG GAT GCC SEQ ID NO: 121 Sense strand: 5′- ACT GTG TA-3′ (SEQ ID AAUCAGGAUGCCACUGUGUA UU-3′ NO: 598) (Position in SEQ ID NO: 442 Antisense strand: 5′- gene sequence: 521) UACACAGUGGCAUCCUGAUU UU-3′ SEQ ID NO: 274 dsRNA: 5′-AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3′ Target sequence 122: 5′- GC content: 40.9% CTG GAT GAG AAG SEQ ID NO: 122 Sense strand: 5′- GTT AGT GA-3′ (SEQ ID CUGGAUGAGAAGGUUAGUGA UU-3′ NO: 599) (Position in SEQ ID NO: 443 Antisense strand: 5′- gene sequence: 551) UCACUAACCUUCUCAUCCAG
  • the siRNA or dsRNA of the present invention may be one capable of being loaded on a carrier while carrying RNA molecules depending on types of the carrier, which is not particularly limited as long as it is known in the art, and may include, but is not limited to, at least one selected from the group consisting of, for example, liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin, lipid particles, polyethylene glycol, poly-L-guluronic al
  • the siRNA or dsRNA of the present invention may be loaded on porous silica particles, wherein the particles are particles of silica material (SiO 2 ) and have a nano-sized particle diameter.
  • the porous silica particles may be porous particles having nano-sized pores and may carry physiologically active substances (“bioactive materials”) such as siRNA or dsRNA of the present invention on the surfaces of the particles and/or insides of the pores.
  • bioactive materials such as siRNA or dsRNA of the present invention
  • the porous silica particles are biodegradable particles and, when the particles loaded with the bioactive material and is administered in a body, may release the bioactive material while being biodegraded in the body. That is, biodegradation of the porous silica particles results in release of the bioactive material.
  • the porous silica particles according to the present invention may be slowly degraded in the body so that the loaded bioactive material can have sustained release properties.
  • t when a ratio of absorbance in the following Equation 1 becomes 1 ⁇ 2 may be 20 or more.
  • a 0 absorbance of the porous silica particles measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particles into a cylindrical dialysis membrane having pores with a diameter of 50 kDa
  • pH of the suspension is 7.4.
  • a t is absorbance of the porous silica particles measured after t hours elapses from the measurement of A 0 ).
  • Equation 1 indicates how fast the porous silica particles are degraded under environments similar to the body.
  • the absorbance Ao and At may be measured, for example, after placing the porous silica particles and the suspension in a cylindrical dialysis membrane, and further placing the same suspension on the outside of the dialysis membrane.
  • the particles are biodegradable and may be slowly degraded in the suspension, wherein the diameter of 50 kDa corresponds to about 5 nm, the biodegraded particles can pass through a 50 kDa dialysis membrane, this cylindrical dialysis membrane is under horizontal agitation at 60 rpm such that the suspension is evenly admixed, and the degraded particles may come out of the dialysis membrane.
  • the absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the dialysis membrane is replaced with a new suspension.
  • the suspension may be one that is constantly replaced, one that is replaced at a constant period wherein the constant period may be periodic or irregular.
  • the replacement may be performed within a range of 1 hour to 1 week, in particular, at 1-, 2-, 3-, 6-, 12-, 24-hours intervals, or 2-, 3-, 4-, 7-days interval, etc., 2 0 but it is not limited thereto.
  • a ratio of absorbance of 1 ⁇ 2 means that, after t hours, the absorbance becomes half of the initial absorbance, therefore, means that approximately half of the porous silica particles have been degraded.
  • the suspension may be a buffer solution and, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • t when the ratio of absorbance in Equation 1 becomes 1 ⁇ 2 is 20 or more or 24 or more, for example, t may be 20 to 120, specifically, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65, etc. within the above range, but it is not limited thereto.
  • the particles are characterized in that t when the ratio of absorbance in Equation 1 becomes 1 ⁇ 5 may be, for example, 70 to 140, specifically, 80 to 140, 80 to 120, 80 to 110, 70 to 140, 70 to 120, 70 to 110, etc. within the above range, but it is not limited thereto.
  • the particles are characterized in that t when the ratio of absorbance in Equation 1 becomes 1/20 may be, for example, 130 to 220, specifically, 130 to 200, 140 to 200, 140 to 180, 150 to 180, etc. within the above range, but it is not limited thereto.
  • the particles are characterized in that t when the measured absorbance becomes 0.01 or less may be, for example, 250 or more, specifically, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, etc. within the above range while having an upper limit of 2000, but it is not limited thereto.
  • the particles are characterized in that the absorbance ratio in Equation 1 has high positive correlation with t, specifically, Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more, 0.95 or more, etc.
  • Equation 1 means how fast the porous silica particles are degraded under environments similar to the body, for example, may be controlled by adjusting the surface area, particle diameter, pore diameter, substituent on the surface of the porous silica particle and/or inside the pore, compactness of the surface, etc.
  • t may be reduced by increasing the surface area of the particle or may be increased by reducing the surface area thereof.
  • the surface area may be regulated by adjusting the diameter of the particles and/or the diameter of the pores.
  • placing a substituent on the surface of the particle and/or the inside of the pore may reduce direct exposure of the porous silica particles to the environment (such as a solvent), thereby increasing t.
  • loading the bioactive material on the porous silica particles and increasing affinity between the bioactive material and the porous silica particles may reduce direct exposure of the porous silica particles to the environment, thereby increasing t.
  • the surface may be made more densely in the preparation of the particles so as to increase t.
  • the porous silica particles may be, for example, spherical particles, but it is not limited thereto.
  • the average diameter of the porous silica particles may be, for example, 100 to 1000 nm, specifically, 100 to 800 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, 100 to 200 nm, etc. within the above range, but it is not limited thereto.
  • the average pore diameter of the particles may be, for example, 1 to 100 nm, specifically, 4 to 100 nm, 4 to 50 nm, 4 to 30 nm, 10 to 30 nm, etc. within the above range, but it is not limited thereto. Due to the large pore diameter, the particles may carry a large amount of the bioactive material and/or the bioactive material having a large size.
  • the porous silica particles may have a BET surface area of, for example, 200 to 700 m 2 /g, specifically, 200 to 700 m 2 /g, 200 to 650 m 2 /g, 250 to 650 m 2 /g, 300 to 700 m 2 /g, 300 to 650 m 2 /g, 300 to 600 m 2 /g, 300 to 550 m 2 /g, 300 to 500 m 2 /g, 300 to 450 m 2 /g, etc. within the above range, but it is not limited thereto.
  • the porous silica particles may have a volume per gram (g) of, for example, 0.7 to 2.2 ml, specifically, 0.7 to 2.0 ml, 0.8 to 2.2 ml, 0.8 to 2.0 ml, 0.9 to 2.0 ml, 1.0 to 2.0 ml, etc. within the above range, but it is not limited thereto. If the volume per gram (g) is too small, a degradation rate may be too high. Further, it may be difficult to manufacture excessively large particles or the particles may not have a complete shape.
  • the porous silica particles may have a hydrophilic substituent and/or a hydrophobic substituent on an outer surface thereof and/or inside the pore.
  • a hydrophilic substituent and/or a hydrophobic substituent on an outer surface thereof and/or inside the pore.
  • hydrophilic substituents or hydrophobic substituents may be present on both the surface of the particle and the inside of the pore, hydrophilic substituents or hydrophobic substituents may be present on either the surface of the particle or the inside of the pore, otherwise, a hydrophilic substituent may be present on the surface of the particle while a hydrophobic substituent may be present inside of the pore, and vice versa.
  • Release of the bioactive material loaded on the porous silica particles is mainly conducted by degradation of the particles. Specifically, interaction of the porous silica particles with respect to the release environment of the bioactive material is controlled by adjusting the substituent, thereby regulating a degradation rate of the particles thus to control a release rate of the bioactive material. Further, the bioactive material may be diffused and released from the particles wherein adjusting the substituent may regulate a binding force of the bioactive material with the particles, thereby controlling the release of the bioactive material.
  • an additional process may be further included so that a hydrophobic substituent is present inside the pore while a hydrophilic substituent is present on the surface of the particle, in consideration of easiness in use and formulation of the composition according to the present invention.
  • the hydrophilic substituent may include, for example, aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate, polyethylene glycol, substituted or unsubstituted C 1 to C 30 alkyl, substituted or unsubstituted C 3 to C 30 cycloalkyl, substituted or unsubstituted C 6 to C 30 aryl, and C 1 to C 30 ester groups, etc., while the hydrophobic substituent may include, for example, substituted or unsubstituted C 1 to C 30 alkyl, substituted or unsubstituted C 3 to C 30 cycloalkyl, substituted or unsubstituted C 6
  • the “substituted” functional group in the “substituted or unsubstituted” mentioned above may include at least one selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate and polyethylene glycol.
  • the porous silica particles may be positively and/or negatively charged on the outer surface thereof and/ or the inside of the pore.
  • both the surface of the particle and the inside of the pore may be positively or negatively charged.
  • only the surface of the particle or the inside of the pore may be positively or negatively charged. Otherwise, the surface of the particle may be positively charged while the inside of the pore may be negatively charged, and vice versa.
  • the charging may be performed, for example, by the presence of a cationic substituent or an anionic substituent.
  • the cationic substituent may include, for example, an amino group or other nitrogen-containing group as a basic group, while the anionic substituent may include, for example, a carboxyl group (—COOH), a sulfonic acid group (—SO 3 H), or a thiol group (—SH), etc., but it is not limited thereto.
  • interaction between the porous silica particles with respect to the environment for releasing the bioactive material is controlled by adjusting the substituent so that a degradation rate of the particles may be regulated thus to control a release rate of the bioactive material.
  • the bioactive material may be diffused and released from the particles wherein adjusting the substituent may regulate a binding force of the bioactive material with the particles, thereby controlling the release of the bioactive material.
  • the porous silica particles may further include another substituent, which is present on the surface of the particle and/or the inside of the pore, in order to carry a bioactive material, transfer the bioactive material to a target cell, carry a material used for other purposes or bind other additional substituents, etc., wherein the substituent may further include an antibody, a ligand, a cell permeable peptide, an aptamer, etc. coupled thereto.
  • the surface modification may be performed, for example, by reacting a compound having a substituent to be introduced, with the particles.
  • the compound may include, for example, alkoxysilane having a Cl to C10 alkoxy group, but it is not limited thereto.
  • the alkoxysilane may have at least one alkoxy group, specifically, 1 to 3 alkoxy groups, and may have a substituent to be introduced into a site in which the alkoxy group is not bonded or a substituent substituted with the alkoxy group.
  • the porous silica particles may be prepared by, for example, a small pore particle preparation and pore expansion process. If necessary, the particles may be prepared through further calcination, and surface modification processes, etc. If the particles are subjected to both the calcination and the surface modification processes, the particles may be surface-modified after the calcinations.
  • the small pore particles may be, for example, particles having an average pore diameter of 1 to 5 nm.
  • the small pore particles may be obtained by adding a surfactant and a silica precursor to a solvent and then agitating and homogenizing the solution.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides
  • a ratio of water and an organic solvent may be used in a volume ratio of, for example, 1:0.7 to 1.5, e.g., 1:0.8 to 1.3, but it is not limited thereto.
  • the surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added in an amount of, for example, 1 to 10 g per liter of solvent, specifically, 1 to 8 g, 2 to 8 g, 3 to 8 g, etc. within the above range, but it is not limited thereto.
  • the silica precursor may be added after the agitation with addition of the surfactant to the solvent.
  • the silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but it is not limited thereto.
  • the agitation may be performed, for example, for 10 to 30 minutes, but it is not limited thereto.
  • the silica precursor may be added thereto, for example, in an amount of 0.5 to 5 ml per liter of solvent, specifically, 0.5 to 4 ml, 0.5 to 3 ml, 0.5 to 2 ml, 1 to 2 ml, etc. within the above range, but it is not limited thereto. Rather, if necessary, sodium hydroxide as a catalyst may be further used, wherein the catalyst may be added while agitating after adding the surfactant to the solvent and before adding the silica precursor to the solvent.
  • Sodium hydroxide may be used in an amount of, for example, 0.5 to 8 ml per liter of solvent, specifically, 0.5 to 5 ml, 0.5 to 4 ml, 1 to 4 ml, 1 to 3 ml, 2 to 3 ml, etc. within the above range, based on 1 M aqueous sodium hydroxide solution, but is not limited thereto.
  • the solution may be reacted with agitation.
  • the agitation may be performed, for example, for 2 to 15 hours, specifically, 3 to 15 hours, 4 to 15 hours, 4 to 13 hours, 5 to 12 hours, 6 to 12 hours, 6 to 10 hours, etc. within the above range, but it is not limited thereto. If an agitating time (reaction time) is too short, nucleation may be insufficient.
  • the solution may be aged. Aging may be performed, for example, for 8 to 24 hours, specifically, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours, 10 to 14 hours, etc. within the above range, but it is not limited thereto.
  • reaction product may be washed and dried to obtain porous silica particles and, if necessary, unreacted material may be isolated before washing, which may be performed, for example, by separating the supernatant through centrifugation.
  • the centrifugation may be implemented, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing may be carried out with water and/or an organic solvent.
  • water and the organic solvent may be used once or several times by turns.
  • water and/or the organic solvent may be used alone for washing once or several times.
  • Such several times may include, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less, etc.
  • the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides such as N,N-dimethylformamide, N,N
  • the washing may be performed under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing may be performed by filtering particles with a filter without centrifugation.
  • the filter may include pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone.
  • the several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like.
  • the drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • the pores of the obtained porous silica particles may be expanded using, for example, a pore swelling agent.
  • the pore swelling agent used herein may include, for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, etc. and, specifically, trimethylbenzene may be used, but it is not limited thereto.
  • the pore swelling agent used herein may be, for example, N,N-dimethylhexadecylamine (DMHA), but it is not limited thereto.
  • DMHA N,N-dimethylhexadecylamine
  • Pore expansion described above may be performed, for example, by mixing porous silica particles in a solvent with a pore swelling agent, and heating and reacting the mixture.
  • the solvent used herein may be, for example, water and/or an organic solvent.
  • the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol
  • the porous silica particles may be added in a ratio of, for example, 10 to 200 g per liter of solvent, specifically, 10 to 150 g, 10 to 100 g, 30 to 100 g, 40 to 100 g, 50 to 100 g, 50 to 80 g, 60 to 80 g, etc. within the above range, but it is not limited thereto.
  • the porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed therein.
  • the second solvent may be added after the porous silica particles are dispersed in the first solvent.
  • the pore swelling agent may be added in an amount of, for example, 10 to 200 parts by volume (vol. parts), specifically, 100 to 150 vol. parts, 10 to 100 vol. parts, 10 to 80 vol. parts, 30 to 80 vol. parts, 30 to 70 vol. parts based on 100 vol. parts of solvent within the above range, but it is not limited thereto.
  • the reaction may be performed, for example, at 120 to 180° C., specifically, 120 to 170° C., 120 to 160° C., 120 to 150° C., 130 to 180° C., 130 to 170° C., 130 to 160° C., 130 to 150° C., etc. within the above range, but it is not limited thereto.
  • the reaction may be performed, for example, for 24 to 96 hours, 2 0 specifically, 30 to 96 hours, 30 to 80 hours, 30 to 72 hours, 24 to 80 hours, 24 to 72 hours, 36 to 96 hours, 36 to 80 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 48 to 96 hours, 48 to 88 hours, 48 to 80 hours, 48 to 72 hours, etc. within the above range, but it is not limited thereto.
  • the reaction may be performed sufficiently without being too much. For example, when the reaction temperature is lower, the reaction time may be increased, otherwise, when the reaction temperature is lower, the reaction time may be shortened. If the reaction is not sufficient, pore expansion may not be sufficient. On the other hand, if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.
  • the reaction may be performed, for example, while gradually increasing the temperature. Specifically, the reaction may be performed while gradually increasing the temperature at a rate of 0.5 to 15° C./min from the room temperature, specifically, 1 to 15° C./min, 3 to 15° C./min, 3 to 12° C./min, 3 to 10° C./min, etc. within the above range, but it is not limited thereto.
  • the reaction solution may be cooled slowly, for example, cooled by lowering the temperature step by step. Specifically, the reaction solution may be cooled by gradually decreasing the temperature at a rate of 0.5 to 20° C./min to room temperature, specifically, 1 to 20° C./min, 3 to 20° C./min, 3 to 12° C./min, 3 to 10° C./min, etc. within the above range, but it is not limited thereto.
  • reaction product After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores. If necessary, unreacted material may be isolated prior to washing, for example, by centrifugation to separate a supernatant.
  • the centrifugation may be performed, for example, at 6,000 to 10,000 rpm for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing may be carried out with water and/or an organic solvent.
  • water and the organic solvent may be used once or several times by turns.
  • water and/or the organic solvent may be used alone for washing once or several times.
  • Such several times may include, for example, two or more, ten or less, specifically, three times, 4 times, 5 times, 6 times, 7 times, 8 times, etc.
  • the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; specifically, alcohol,
  • the washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing may be performed by filtering particles with a filter without centrifugation.
  • the filter may have pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone.
  • the several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like.
  • the drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • the pores of the obtained porous silica particles may be subjected to calcinations, which is a process of heating the particles to have a denser structure on the surface thereof and the inside of the pore, and removing organic materials filling the pores.
  • the calcinations may be performed at 400 to 700° C. for 3 to 8 hours, specifically, at 500 to 600° C. for 4 to 5 hours, but it is not limited thereto.
  • porous silica particles may be modified on the surface and/or the inside of the pore as described above.
  • the surface modification may be performed on the surface of the particle and/or the inside of the pore.
  • the surface of the particle and the inside of the pore may be surface-modified in the same manner or differently.
  • the particles may be charged or have hydrophilic and/or hydrophobic properties through surface modification.
  • the surface modification may be performed, for example, by reacting a compound having a hydrophilic, hydrophobic, cationic or anionic substituent to be introduced, with the particles.
  • the compound may include, for example, alkoxysilane having a C1 to C10 alkoxy group, but it is not limited thereto.
  • the alkoxysilane may have at least one alkoxy group, specifically, 1 to 3 alkoxy groups, and may have a substituent to be introduced into a site in which the alkoxy group is not bonded or a substituent substituted with the alkoxy group.
  • an alkoxysilane When the alkoxysilane is reacted with the porous silicon particles, an alkoxysilane can be bonded to the surface of the porous silica particle and/or the inside of pore through a covalent bond between a silicon atom and an oxygen atom. Further, since the alkoxysilane has a substituent to be introduced, this substituent may be introduced into the surface of the porous silica particle and/or the inside of the pore.
  • the above reaction may be performed by reacting the porous silica particles dispersed in a solvent with alkoxysilane.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides
  • the positively charging may be performed by reacting the particles with, for example, alkoxysilane having a basic group, that is, a nitrogen-containing group such as amino, aminoalkyl, etc.
  • alkoxysilane having a basic group that is, a nitrogen-containing group such as amino, aminoalkyl, etc.
  • the negatively charging may be performed by reacting the particles with, for example, alkoxysilane having an acidic group such as carboxyl, sulfonic acid, thiol, etc.
  • alkoxysilane having an acidic group such as carboxyl, sulfonic acid, thiol, etc.
  • 3-mercaptopropyl)trimethoxysilane may be used, but it is not limited thereto.
  • the hydrophilic property may be obtained by reacting the particles with, for example, alkoxysilane having a hydrophilic group such as hydroxyl, carboxyl, amino, carbonyl, sulfhydryl, phosphate, thiol, ammonium, ester, imide, thioimide, keto, ether, indene, sulfonyl, polyethyleneglycol, etc.
  • alkoxysilane having a hydrophilic group such as hydroxyl, carboxyl, amino, carbonyl, sulfhydryl, phosphate, thiol, ammonium, ester, imide, thioimide, keto, ether, indene, sulfonyl, polyethyleneglycol, etc.
  • N-[3-(trimethoxysilyl) propyl]ethylenediamine, N1-(3-trimethoxysilylpropyl)diethylenetriamine, (3-aminopropyl)trimethoxysilane, (3-mercaptopropyl)trimethoxysilane, trimethoxy [3-(methylamino)propyl]silane, 3-(2-aminoethylamino)propyldimethoxymethylsilane, etc. may be used, but it is not limited thereto.
  • the hydrophobic property may be obtained by reacting the particles with, for example, alkoxysilane having a hydrophobic substituent such as substituted or unsubstituted C 1 C 30 alkyl, substituted or unsubstituted C 3 to C 30 cycloalkyl, substituted or unsubstituted C 6 to C 30 aryl, substituted or unsubstituted C 2 to C 30 heteroaryl, halogen, C 1 to C 30 ester, and halogen-containing groups, etc.
  • alkoxysilane having a hydrophobic substituent such as substituted or unsubstituted C 1 C 30 alkyl, substituted or unsubstituted C 3 to C 30 cycloalkyl, substituted or unsubstituted C 6 to C 30 aryl, substituted or unsubstituted C 2 to C 30 heteroaryl, halogen, C 1 to C 30 ester, and halogen-containing groups, etc.
  • an additional process may be further included so that a hydrophobic substituent is present inside the pore while a hydrophilic substituent is present on the surface of the particle, in consideration of easiness in use and formulation of the composition according to the present invention.
  • a substituent for binding another material other than the bioactive material to the surface of the particle may be further provided.
  • the surface modification may be performed in combination.
  • two or more surface modification may be performed on the outer surface of the particle or inside the pore.
  • a compound containing a carboxyl group may be bonded to an amide-introduced silica particle through amide bond to change positively charged particles to have different surface characteristics, but it is not limited thereto.
  • the reaction of the porous silica particles with alkoxysilane may be performed, for example, under heating.
  • the heating may be performed at 80 to 180° C., for example, in a range of 80 to 160° C., 80 to 150° C., 100 to 160° C., 100 to 150° C., 110 to 150° C., etc., but it is not limited thereto.
  • the reaction of the particles with alkoxysilane may be implemented for 4 to 20 hours, for example, in a range of 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 14 hours, etc., but it is not limited thereto.
  • a reaction temperature, time, and the amount of the compound used for surface modification may be selected depending on a desired extent of surface modification. Further, varying reaction conditions depending on hydrophilicity, hydrophobicity and a charge level of the bioactive material may regulate hydrophilicity, hydrophobicity and charge level of the silica particles, thereby controlling the release rate of the bioactive material. For example, if the bioactive material has strong negative charge at neutral pH, the reaction temperature may be increased, the reaction time may be extended, or an amount of the compound to be treated may also be increased so that the porous silica particles have strong positive charge, but it is not limited thereto.
  • porous silica particles may be manufactured by, for example, preparation of small pore particles, expansion of pores, surface modification, modification of inside of the pore and the like.
  • Preparation of the small pore particles and pore expansion may be performed by the processes described above, and the washing and drying processes may be performed after the preparation of the small pore particles and after the pore expansion.
  • isolation of the unreacted material may be preceded by washing, for example, conducted by separating the supernatant through centrifugation.
  • the centrifugation may be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing after the preparation of the small pore particles may be performed by any method under conditions within the above-illustrated range, but it is not limited thereto.
  • washing after the pore expansion may be performed under more relaxed conditions than the above illustrative embodiments.
  • washing may be carried out three times or less, but it is not limited thereto.
  • the surface of the particle and/or the inside of the pore may be modified by the above-described method, wherein the modification may be performed in an order of the surface of the particle and then the inside of the pore, and particle washing may be further performed between the above two processes.
  • the pores are filled with a reaction solution such as a surfactant used in the particle preparation and the pore expansion, such that the inside of the pore is not modified during surface modification, instead, only the surface of the particle may be modified. After then, washing the particles may remove the reaction solution in the pores.
  • a reaction solution such as a surfactant used in the particle preparation and the pore expansion
  • Particle washing between surface modification and modification of the inside of the pore may be performed with water and/or an organic solvent.
  • water and the organic solvent may be used once or several times by turns.
  • water and/or the organic solvent may be used alone for washing once or several times. Such several times may include, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less, etc.
  • the washing may be performed under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • the washing may be performed by filtering particles with a filter without centrifugation.
  • the filter may include pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone. The several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like.
  • the drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • the bioactive material such as siRNA or dsRNA of the present invention may be loaded on the surface of the particle and/or the inside of the pore.
  • the loading may be implemented, for example, by mixing the porous silica particles and the bioactive material in a solvent.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propan
  • a phosphate buffered saline solution PBS
  • simulated body fluid SBF
  • borate-buffered saline tris-buffered saline
  • a ratio of the porous silica particles and the bioactive material is not particularly limited and, for example, the weight ratio may be 1:0.05 to 0.8, specifically, 1:0.05 to 0.7, 1:0.05 to 0.6, 1:0.1 to 0.8, 1:0.1 to 0.6, 1:0.2 to 0.8, 1:0.2 to 0.6, etc. within the above range.
  • the bioactive material such as siRNA or dsRNA of the present invention loaded on the porous silica particles may be gradually released over an extended period of time. Such slow release may be continuous or non-continuous, or linear or non-linear, and may vary due to the characteristics of the porous silica particles and/or interaction thereof with the bioactive material.
  • the bioactive material loaded on the porous silica particles is released while the porous silica particles are biodegraded. More particularly, the porous silica particles according to the present invention may be slowly degraded to release the loaded bioactive material in a sustained manner. Such release may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface of the particle and/or the inside of pore, compactness of the porous silica particles, and the like, but it is not limited thereto.
  • the bioactive material loaded on the particles may be released while being separated from the porous silica particles and diffused, which is affected by the relationship between the porous silica particles, the bioactive material and the bioactive material releasing environment. Therefore, adjusting these conditions may control the release of bioactive material. For example, the release of bioactive material may be controlled by strengthening or weakening the binding force of the porous silica particles with the bioactive material by surface modification.
  • the surface of the particle and/or the inside of the pore may have a hydrophobic substituent to increase the binding force between the particles and the bioactive material, whereby the bioactive material may be released in a sustained manner.
  • This may be achieved by, for example, surface modification of the particles with alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” means being insoluble (practically insoluble) or only slightly soluble (with respect to water), which is a terminology defined in “pharmaceutical Science” 18 th Edition (U.S.P, Remington, Mack Publishing Company).
  • the poorly water-soluble bioactive material may have, for example, water solubility of less than 10 g/L, specifically less than 5 g/L, more specifically less than 1 g/L at 1 atmosphere and 25° C., but it is not limited thereto.
  • the surface of the particle and/or the inside of the pore may have a hydrophilic substituent to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive materials may be released in a sustained manner.
  • the water-soluble bioactive material may have, for example, water solubility of 10 g/L or more at 1 atmosphere and 25° C., but it is not limited thereto.
  • the surface of the particle and/or the inside of the pore may be charged with the opposite charge thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be released in a sustained manner.
  • This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having an acidic group or a basic group.
  • the surface of the particle and/or the inside of the pore may be negatively charged at neutral pH thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be released in a sustained manner.
  • This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having an acidic group such as a carboxyl group (-COOH), sulfonic acid group (—SO 3 H), etc.
  • the surface of the particle and/or the inside of the pore may be positively charged thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be release in a sustained manner.
  • This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having a basic group such as an amino group, nitrogen-containing group, etc.
  • the loaded bioactive material may be released for a period of, for example, 7 days to 1 year or more depending on the type of treatment required, release environment, and porous silica particles to be used, etc.
  • porous silica particles are biodegradable and may be degraded by 100%, the bioactive material loaded thereon can be released by 100%.
  • the pharmaceutical composition for preventing or treating liver cancer which includes siRNA or dsRNA of the present invention, may further include pharmaceutically acceptable carrier and may be formulated together with the same.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate an organism and does not inhibit biological activities and properties of the administered compound.
  • the pharmaceutically acceptable carrier in a composition formulated in a liquid solution is sterile and physiologically compatible, and may include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a combination of one or more of these components.
  • compositions may also be added thereto.
  • additives such as antioxidants, buffers and bacteriostatic agents may also be added thereto.
  • diluents, dispersants, surfactants, binders and lubricants may also be added so as to formulate the composition into injectable formulations such as aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets and the like.
  • composition of the present invention is applicable in any type of formulation that contains the siRNA or dsRNA of the present invention as an active ingredient, and may be prepared in oral or parenteral formulations.
  • Such pharmaceutical formulations of the invention may include any one suitable for oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous) administration, or otherwise, may be suitable for administration through inhalation or insufflation.
  • composition of the present invention may be administered in a pharmaceutically effective amount.
  • An effective dose level may be determined in consideration of the type of disease, severity, activity of the drug, sensitivity to the drug, administration time, administration route and rate of release, duration of treatment, factors including concurrent drug use, and other factors well known in the medical field.
  • the composition of the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer a minimum amount that can obtain maximum effects without side effects, which can be easily determined by those skilled in the art.
  • Dosage of the composition of the present invention may vary greatly depending on a weight, age, gender and/or health condition of a patient, diet, administration time, method of administration, excretion rate and severity of the disease.
  • an appropriate dosage may depend on the amount of drug accumulated in the body and/or specific efficacy of the siRNA or dsRNA of the present invention to be used.
  • the dosage may be estimated based on EC50 determined to be effective in in vivo animal models as well as in vitro.
  • the dosage may range from 0.01 ⁇ g to 1 g per kg of body weight, and the composition may be administered once or several times per unit period, in daily, weekly, monthly or yearly unit periods.
  • the composition may be continuously administered for a long period of time via an infusion pump.
  • the number of repeated doses is determined in consideration of a retention time of drug remaining in the body, a concentration of drug in the body and the like. Even after the treatment in the course of the disease treatment, the composition may be administered for preventing relapse.
  • composition of the present invention may further include at least one active ingredient having the same or similar function in relation to treatment of liver cancer or a compound which maintains/increases solubility and/or absorbency of the active ingredient.
  • chemotherapeutic agents, anti-inflammatory agents, antiviral agents and/or immune-modulators, etc. may be optionally included.
  • composition of the present invention may be formulated by any conventional method known in the art to provide rapid, sustained or delayed release of the active ingredient after the administration thereof to a mammal.
  • the formulation may be in a form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7 liver cancer cell line were obtained from Korean Cell Line Bank (Seoul, Korea). All of the cell lines were cultured in EMEM (American Type Culture Collection, Manassas, Va.), RPMI-1640 or DMEM medium (Lonza, Walkersville, Md.) which is supplemented with 10% fetal bovine serum (FBS, Lonza) and 100 units/mL penicillin-streptomycin
  • siRNA and dsRNA used in this experiment were synthesized by
  • Genomic DNA from tissues and cells was isolated using DNAzol reagent (Invitrogen) according to the manual.
  • DNAzol reagent Invitrogen
  • the SF3B4 genomic DNA region was amplified from 20 pairs (non-tumor and tumor) HCC tissues using a primer set from exon-1 to intron-1 according to the genome sequence of Genbank accession No. NC_000001.11.
  • qRT-PCR was performed as described above, and glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous loading control.
  • the primer sequences used for RT-PCR and qRT-PCR are shown in Table 5 below.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • Transfected cells were seeded in wells of a 6-well plate. At 100% confluence, a scratch was made on a uniform layer using a micropipette tip. Photographs of the same area of the wound were taken after 0 and 24 h with IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).
  • xenograft tumorigenesis assay 1 ⁇ 10 7 cells of transfected cells were mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v) Matrigel matrix (BD Biosciences). Cell suspensions were subcutaneously injected in 6-week-old male Balb/c-nude mice. Mice were examined twice per week for tumor formation at the injection sites. Tumor volumes were calculated using: 0.5 ⁇ length (L) ⁇ width (W 2 ). Each experimental group consisted of 10 mice and tumor growth was quantified by measuring tumor sizes in three orthogonal direction using calipers. Results are expressed as mean tumor volumes and 95% confidence interval. The H-ras12V activated homozygous transgenic mice were kindly provided by Dr.
  • Dae-Yeoul Yu (Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, Daej eon, Korea).
  • Transgenic mice were H-ras12V activated. Male mice spontaneously developed HCC beginning at 15-weeks-of-age. We surgically obtained the non-tumor region and HCC mass from five mice (35-weeks-old) and pick out three pairs of HCC tissue by pathological scoring. Diethylnitrosamine (DEN) was used to induce HCC.
  • DEN Diethylnitrosamine
  • siRNA specific to BANF1, PLOD3, SF3B4 was loaded in 80 ⁇ l of 3 nmol InViVojectionTM RNAi-nano reagent (the porous silica nanoparticles in EXAMPLE 1-12(1)-2)-(ii), Cat. No. DHMSN-vivoRNA; Lemonex Inc., Seoul, Korea) and prepared in 200 ⁇ l of PBS.
  • a mixture of siRNA or dsRNA and nanoparticles was intravenously injected into H-ras transgenic HCC mouse model through tail vein every week from week 9 to week 23. Sonograms (ultrasonic photographs) were taken at 17, 19, and 21 weeks by an ultrasonic machine (Affiniti 50, Philips, Seoul, Korea).
  • a 2 L round bottom flask was charged with 960 ml of distilled water (DW) and 810 ml of MeOH. 7.88 g of CTAB was added to the flask and 4.52 ml of 1 M NaOH was rapidly added with agitating. The mixture was agitated for 10 minutes to give a homogeneously mixed solution, and 2.6 ml of TMOS was added thereto. The mixture was homogenously mixed under agitation for 6 hours and then aged for 24 hours.
  • reaction solution was centrifuged at 8000 rpm and 25° C. for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C., the product was washed five times with ethanol and distilled water by turns.
  • TMB trimethyl benzene
  • the reaction started at 25° C. and proceeded with heating at a rate of 10° C./min, followed by cooling down at a rate of 1 to 10° C./min in an autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm and 25° C. for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • porous silica particles prepared in the above 2) were placed in a glass vial and heated at 550° C. for 5 hours. After the completion of the reaction, the particles were gradually cooled to room temperature, thereby preparing particles.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that the reaction conditions at the time of pore expansion were changed to 140° C. and 72 hours.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 5-fold larger vessels were used and all the materials were used in a 5-fold capacity.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 920 ml of distilled water and 850 ml of methanol were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 800 ml of distilled water, 1010 ml of methanol and 10.6 g of CTAB were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 620 ml of distilled water, 1380 ml of methanol and 7.88 g of CTAB were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 2.5 ml of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 4.5 ml of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 11 ml of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 12.5 ml of TMB was used at the time of pore expansion.
  • the small pore particles were reacted with TMB in the same manner as in EXAMPLE 1-11(1)-2), cooled, and centrifuged to remove the supernatant. Thereafter, the mixture was centrifuged under the same conditions as in EXAMPLE 1-11(1)-2), washed three times with ethanol and distilled water by turns, and then dried under the same conditions as in EXAMPLE 1-11(1)-2), thereby obtaining powdery silica particles (pore diameter: 10 to 15 nm, particle diameter: 200 nm).
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • porous silica particles in EXAMPLE 1-11(4) were reacted with (3-aminopropyl)triethoxysilane (APTES) thus to be positively charged.
  • APTES (3-aminopropyl)triethoxysilane
  • porous silica particles were dispersed in 10 ml of toluene in a 100 ml round bottom flask provided with a bath sonicator. Then, 1 ml of APTES was added thereto, and the mixture was reacted for 12 hours with agitation at 400 rpm and 130° C.
  • the mixture was slowly cooled to room temperature, centrifuged at 8000 rpm for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • the resultant product was dried in an oven at 70° C. to obtain powdery porous silica particles having an amino group on the surfaces of the particles and the insides of pores.
  • porous silica particles in EXAMPLE 1-11(1) were reacted with trimethoxy(propyl)silane to introduce a propyl group into the surfaces of the particles and the insides of the pores, and then were modified in the same manner as in EXAMPLE 1-12(1), except that 0.35 ml of trimethoxy(propyl)silane was added instead of APTES and the reaction was conducted for 12 hours.
  • porous silica particles of EXAMPLE 1-11(1) were reacted with trimethoxy-n-octylsilane to introduce a propyl group into the surfaces of the particles and the insides of the pores, and then were modified in the same manner as in EXAMPLE 1-12(1), except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and the reaction was conducted for 12 hours.
  • porous silica particles in EXAMPLE 1-11(1) were reacted with succinic anhydride thus to be negatively charged, and then were modified in the same manner as in EXAMPLE 1-12(1)-1), except that dimethyl sulfoxide (DMSO) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES, followed by agitation at room temperature for 24 hours to conduct the reaction, and DMSO was used instead of distilled water at the time of washing.
  • DMSO dimethyl sulfoxide
  • indicator genes of siRNA and dsRNA of the present invention (BANF1 variant 1, BANF1 variant 2, PLOD3, and SF3B4) were analyzed, and the results are shown in Tables 7 to 10 below.
  • siRNAs each of which includes a sense RNA having a sequence shown in Table 11 below and an antisense RNA having a complementary sequence thereto, were subjected to in vitro transfection by the methods described in EXAMPLE 1-2 or 1-8, respectively. Then, expression levels of the corresponding markers of the above siRNAs were measured by Western blotting, and the results are shown in FIG. 1 .
  • FIG. 1 Target gene SEQ ID NO: 311 5′- Banf1 Mouse BANF1 gene CCUCAGCGUUUCAAU CUUUU-3′ SEQ ID NO: 312 5′- Plod3 Mouse PLOD3 gene CGACUGCAGAAUCUC CUCUUU-3′ SEQ ID NO: 313 5′- Sf3b4 Mouse SF3B4 gene CUGCUUUACGAUACU UUCAUU-3′ SEQ ID NO: 314 5′- Control — CCUACGCCACCAAUU UCGU-3′ SEQ ID NO: 28 5′- BANF1 Human BANF1 gene AAGAAGCUGGAGGAA AGGGGUUU-3′ SEQ ID NO: 119 5′- PLOD3 Human PLOD3 gene GCAUCUGGAGCUUUC UGUA UU-3′ SEQ ID NO: 136 5′- SF3B4 Human SF3B gene GCAGUACCUCUGUAA CCGU U
  • siRNAs each of which includes a sense RNA having a sequence shown in Table 12 below and an antisense RNA having a complementary sequence thereto, were subjected to in vitro transfection by the method described in EXAMPLE 1-2. Migration and invasion of markers corresponding to the above siRNAs were analyzed by the method in EXAMPLE 1-5, while a scratch wound healing ability was analyzed by the method in EXAMPLE 1-6, and the analyzed results are shown in FIG. 2 .
  • siRNAs each of which includes a sense RNA having the sequence shown in Table 12 above and an antisense RNA having the complementary sequence thereto, were subjected to in vitro transfection to the SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2. Then, expression levels of the markers corresponding to the siRNAs and expression levels of the EMT regulatory proteins above were analyzed by the method in EXAMPLE 1-9, and the analyzed results are shown in A of FIG. 3 .
  • EMT epithelial-mesenchymal transition
  • the cells with the markers which were knockdown by transfection of siRNAs listed in Table 12, exhibited that the expression levels of N-cadherin, Fibronectin, Snail and Slug, which are epithelial-mesenchymal transition (EMT) regulatory proteins, as well as the expression levels of the markers are inhibited.
  • EMT epithelial-mesenchymal transition
  • siRNAs each of which includes a sense RNA having the sequence shown in Table 12 above and an antisense RNA having the complementary sequence thereto, were subjected to in vitro transfection to the SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2. Thereafter, athymic nude mice were subjected to subcutaneous injection of the transfected cells, followed by analyzing sizes of hepatic tumors and survival rates of the mice, and the analyzed results are shown in B of FIG. 3 .
  • knockdown of the markers by the siRNA or dsRNA of the present invention may reduce overall tumor growth rate and decrease the average tumor volume.
  • the tumor-free survival rates of the experimental groups are significantly higher than that of the control group. Specifically, when 50 days elapses after the subcutaneous injection of the transfected cells, the control group exhibited tumors in 6 mice of 10 mice whereas the experimental group exhibited tumors in 1 to 2 mice only among 10 mice, thereby indicating that the siRNA or dsRNA of the present invention may effectively inhibit the growth of hepatic tumors.
  • siRNAs each of which includes a sense RNA having a sequence shown in Table 13 below and an antisense RNA having a complementary sequence thereto, were subjected to in vivo transfection by the method described in EXAMPLE 1-8, and processes thereof, ultrasonic images and the number of tumors over time are shown in A of FIG. 4 . Further, expression inhibitory levels for the indicator genes by the siRNAs loaded on the porous nanoparticles were analyzed by the method in EXAMPLE 1-9 and shown in B of FIG. 4 .
  • FIG. 1 Target gene SEQ ID 5′- Banfl Mouse BANF1 NO: 311 CCUCAGCGUUUCAAUCU gene UUUU-3′ SEQ ID 5′- Plod3 Mouse PLOD3 NO: 312 CGACUGCAGAAUCUCCU gene CUUU-3′ SEQ ID 5′- 5f3b4 Mouse 5F3B4 NO: 313 CUGCUUUACGAUACUUU gene CAUU-3′
  • the small pore particles and the prepared porous silica particles in EXAMPLE 1-11(1) to (3) were observed with a microscope to identify whether the small pore particles were uniformly formed and the pores were sufficiently expanded thus to uniformly form the porous silica particle ( FIGS. 5 to 8 ).
  • FIG. 5 is photographs illustrating the porous silica particles in EXAMPLE 1-11(1)
  • FIG. 6 is photographs illustrating the porous silica particles in EXAMPLE 1-11(2), demonstrating that the porous silica particles in a spherical shape with sufficiently expanded pores were uniformly formed.
  • FIG. 7 is photographs illustrating the small pore particles in EXAMPLE 1-11(1)
  • FIG. 8 is comparative photographs illustrating the small pore particles in EXAMPLEs 1-11(1) and 1-11(3), demonstrating that spherical small pore particles were uniformly formed.
  • porous silica particles were almost completely degraded after 360 hours elapse from the observation.
  • a 0 absorbance of the porous silica particles measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particles into a cylindrical dialysis membrane having pores with a diameter of 50 kDa
  • porous silica particle powders were dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a dialysis membrane having a pore diameter of 50 kDa shown in FIG. 10 . 15 ml of SBF was added to an outer membrane, and SBF of the outer membrane was replaced every 12 hours. Degradation of the porous silica particles was performed while horizontally agitating at 37° C. and 60 rpm.
  • the absorbance ratio of the porous silica particles in EXAMPLE 1-11(1) was 2 5 measured according to the above method, and the results are shown in FIG. 11 .
  • porous silica particles in the examples have significantly larger t than the control.
  • the absorbance of the porous silica particles in EXAMPLE 1-11(4) was measured by pH.
  • the absorbance was measured in SBF and in Tris at pH 2, 5 and 7.4, respectively, and the results are shown in FIG. 14 .
  • the release solvent was recovered at 0.5, 1, 2, 4, 8, 12, 24 hours prior to 24 hours and then, every 24 hours, 0.5 ml of the release solvent was recovered for fluorescence measurement and SBF was added thereto.
  • the time at which 50% of the siRNA was released was about 40 hours or more.
  • siRNAs of the present invention can play a role of a transporter in a desired level for study of siRNA delivery in animal level. tumor inhibitory rates due to the release of bioactive material in mice (rats) were confirmed.
  • mice Balb/c nude male mice (5 weeks old) were purchased from Orient Bio Inc., and 3 million HeLa cells (cervical cancer cells) were dispersed in sterilized 1 ⁇ PBS to proliferate Xenograft tumors subcutaneously injected into the mice.
  • PBS FITC-porous silica particles
  • FITC-porous silica particles loaded with siRNA porous silica particles in EXAMPLE 1-12(1)-2)-(ii)
  • FITC labeling were implemented by: dispersing 50 mg of silica particles in 1 ml of dimethyl sulfoxide (DMSO); adding 25 ⁇ g (10 ⁇ l) of FITC-NHS (N-hydroxysuccinimide) solution (2.5 mg/mL) thereto; reacting the mixture at room temperature for 18 hours while shielding light with aluminum foil; purifying the reaction product through centrifugation (8500 rpm, 10 minutes); discarding the supernatant while collecting settled particles; and evenly dispersing the particles in ethanol, wherein the above processes were repeated three and four times with ethanol and distilled water to purify until FITC color is invisible in the supernatant.
  • DMSO dimethyl sulfoxide
  • control group refers to administration of PBS alone
  • Cy5-siRNA refers to administration of siRNA in EXAMPLE 1-13 alone
  • FITC-DDV refers to administration of FITC-labeled porous silica particles alone
  • the complex refers to administration of FITC-labeled porous silica particles loaded with the siRNA in EXAMPLE 1-13.

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Abstract

The present invention provides siRNA or dsRNA, which can effectively inhibit the expression of three highly expressed markers in liver cancer, and a pharmaceutical composition including the same can obtain excellent effects of preventing or treating liver cancer through RNAi. A pharmaceutical composition for preventing or treating liver cancer according to an embodiment of the present invention includes at least one of siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, and an antisense RNA having a complementary sequence thereto and dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY
  • This application claims benefit under 35 U.S.C. 119(e), 120, 121, or 365(c), and is a National Stage Entry from International Application No. PCT/KR2018/008611, filed on Jul. 30, 2018, which claims priority to the benefit of U.S. Patent Application No. 62/538,034 filed in the US Patent Office on Jul. 28, 2017 and Korean Patent Application No. 10-2018-0088375 filed in the Korean Intellectual Property Office on Jul. 30, 2018, the entire contents of which are incorporated herein by reference.
    • TECHNICAL FIELD
  • The present invention relates to a pharmaceutical composition for preventing or treating liver cancer.
    • BACKGROUND ART
  • Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. HCC is one of few cancers that have been recently increased in incidence.
  • The primary treatment of HCC is surgical resection, and most of patients are not eligible for curative treatment at the initial treatment stage. Resection and transdermal ablation involve a recurrence rate of 70% after 5 years and thus are closely related to the survival rate.
  • Like other cancers, HCC is also characterized by multiple tumor progression. The damaged liver tissues in the early stage evolve to small nodular hypercellular lesions called dysplastic nodules (DNs). Such pre-cancerous lesion develops into small, well-differentiated hepatocytes with an ambiguous nodular pattern and then progresses to early hepatocellular carcinoma (eHCC), which is defined as progressive HCC characterized by an epileptic appearance and frequent microvascular invasion. Based on current knowledge of an occurrence of multilevel HCC, high-critical patients are closely followed up, and diagnostic images show that a specific lesion with a small size and unknown cause was increased in number. Ultrasound-guided needle biopsy is performed on such lesions. The lesion would be subjected to treatment if it is determined as a cancer by histological diagnosis. However, eHCC generally exhibits minimal dysplasia and lacks clear invasive or destructive growth. Therefore, even for hepatopathologists, it is often difficult to distinguish recurrent nodules, precancerous lesions and early lesions. Due to such reasons, discovery of objective molecular markers that standardize histological diagnosis of eHCC and induce appropriate therapy is eagerly required. In addition, the discovery of biomarkers related to accurate HCC diagnosis may facilitate identification of precancerous lesions possibly progressing to HCC and to determine surgically resectable lesions, thereby supporting a surgeon to design a surgical range in HCC patients. Identifying additional molecular markers that predict possible occurrence of HCC in precancerous lesions may be helpful for identifying patients at risk for recurrence following surgical resection.
  • The present study is intended to establish a gene selection strategy to identify potential causative genes by combining clinicopathological and gene expression data of staged hepatocarcinoma tissues defined by hepatopathologists. As a result, 10 genes expected to be the cause of early stage HCC could be selected.
  • Clinical and experimental studies have demonstrated that barriers to procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 and splicing factor 3b subunit 4 among the 10 presumptive driving genes could indicate HCC in precancerous lesions, and also could diagnose eHCC in a large scale of HCC patients, compared to glypican 3, glutamine synthetase and heat shock protein 70 which are current HCC diagnostic marker trio.
  • In vivo experiments and in vitro tumor formation analysis demonstrated that target destruction of BANF1, PLOD3 and SF3B4 genes inhibits tumor and metastatic characteristics of HCC cells. Excessive response of SFB4 could increase slug in p27 and HCC cells to inhibit epithelial-mesenchymal transition (EMT), which contributes to transformation and proliferation of malignant cells, hence interfering with a cell cycle checkpoint and thus causing over-activation of spliceosome. Further, it could be seen that production of selective splicing variants inhibiting the growth of KLF4 tumor was accelerated.
  • The results described above suggest that novel HCC diagnostic markers, that is, BANF1, PLOD3 and SF3B contribute to early malignant transformation of hepatocytes in formation of hepatic tumor and are also targets for molecular therapy of liver malignancy.
    • SUMMARY
  • It is an object of the present invention to provide a pharmaceutical composition for preventing or treating liver cancer that knockdown a specific gene highly expressed in early stage liver cancer cells.
  • 1 A pharmaceutical composition for preventing or treating liver cancer, including: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.
  • 2 The composition according to the above 1, wherein the composition includes:
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 12, 14 to 19, 21, 23, 24, 26, 28 to 34, 35 to 37, 39 to 41, 43, 45 to 47, 49 to 53, 55 to 60, 62 to 73, 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 165, 167 to 172, 174, 176, 177, 179, 181 to 187, 188 to 190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to 226, 228 to 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310.
  • 3 The composition according to the above 1, wherein the composition includes:
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28, and an antisense
  • RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181.
  • 4. The composition according to the above 1, wherein the composition includes:
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208.
  • 5. The composition according to the above 1, wherein the composition includes:
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273.
  • 6. The composition according to the above 1, wherein the composition includes:
  • siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157, and an antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310.
  • 7. The composition according to any one of the above 1 to 6,
  • wherein the siRNA or dsRNA is loaded on at least one carrier selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin, lipid particles, polyethylene glycol, poly-L-guluronic alginate, polyglycolic-polylactic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and hydrogel.
  • 8. The composition according to the above 7,
  • wherein the carrier is a porous silica particle characterized in that t when a ratio of absorbance in the following Equation 1 becomes ½ is 20 or more:

  • At/A0   [Equation 1]
  • (wherein A0 is absorbance of the porous silica particle measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particle into a cylindrical dialysis membrane having pores with a diameter of 50 kDa,
  • 15 ml of the same solvent as the suspension is placed outside the dialysis membrane while being in contact with the dialysis membrane, followed by horizontal agitation at 60 rpm and 37° C. inside and outside the dialysis membrane, and
  • At is absorbance of the porous silica particle measured after t hours elapses from the measurement of A0).
  • 9. The composition according to the above 8, wherein the t is 40 or more.
  • 10. The composition according to the above 8,
  • wherein the siRNA includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 28, 119 and 136, and an antisense RNA having a complementary sequence thereto, and
  • the dsRNA has at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 181, 272 and 289.
  • 11. The composition according to the above 8,
  • wherein the porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
  • 12. The composition according to the above 8,
  • wherein the porous silica particle has at least one hydrophilic substituent selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate, polyethylene glycol, substituted or unsubstituted C1 to C30 alkyl, substituted or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted C6 to C30 aryl, and C1 to C30 ester groups.
  • 13. The composition of claim 8,
  • wherein the porous silica particle is positively or negatively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
  • 14. The composition of claim 8,
  • wherein the porous silica particle is positively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
  • 15. The composition of claim 8,
  • wherein the porous silica particle has an average particle diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
  • The pharmaceutical composition of the present invention provides preventive and therapeutic effects of liver cancer by specifically knocking down the genes expressed in early stage liver cancer cells, so as to prevent the development of liver cancer and inhibit the metastasis and proliferation of liver cancer cells.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is views illustrating results of measuring expression levels of indicator factors (“markers”) corresponding to siRNAs by Western blotting, respectively, after in vitro transfection of siRNAs to Hepa-1c1c7 and SNU-449 cell lines in EXAMPLE 1 by methods described in EXAMPLE 1-2 or 1-8, wherein each siRNA includes a sense RNA having a sequence in Table 11 and an antisense RNA having a complementary sequence to that of the sense RNA.
  • FIG. 2 is views illustrating results of analyzing migration and invasion of markers corresponding to siRNAs by a method described in EXAMPLE 1-5 and analyzing scratch wound healing ability of the same by a method described in EXAMPLE 1-6, respectively, after in vitro transfection of siRNAs to SNU-449 cell line in EXAMPLE 1-1 by a method described in EXAMPLE 1-2, wherein each siRNA includes a sense RNA having a sequence in Table 12 and an antisense RNA having a complementary sequence to the sequence of the sense RNA.
  • A of FIG. 3 is views illustrating results of analyzing expression levels of markers corresponding to siRNAs and EMT regulatory proteins by a method described in EXAMPLE 1-9, respectively, after in vitro transfection of siRNAs to SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2, wherein each siRNA includes a sense RNA having a sequence in Table 12 and an antisense RNA having a complementary sequence to the sequence of the sense RNA; and B of FIG. 3 is views illustrating the analyzed results of hepatic tumor sizes and survival rates of mice, after subcutaneous injection of transfected cells in the above (A) into athymic nude mice.
  • A of FIG. 4 is views illustrating processes of in vivo transfection of siRNAs by a method described in EXAMPLE 1-8, ultrasonic images and the number of tumors over time, wherein each siRNA includes a sense RNA having a sequence in Table 13 and an antisense RNA having a complementary sequence to the sequence of the sense RNA; and B of FIG. 4 is a view illustrating results of analyzing expression inhibitory levels of siRNAs loaded on porous nanoparticles to indicator genes corresponding to the siRNAs by the method described in EXAMPLE 1-9.
  • FIG. 5 is micrographs illustrating porous silica particles.
  • FIG. 6 is micrographs illustrating porous silica particles.
  • FIG. 7 is micrographs illustrating small pore particles during production of the porous silica particles.
  • FIG. 8 is micrographs illustrating small pore particles.
  • FIG. 9 is micrographs demonstrating biodegradability of porous silica particles.
  • FIG. 10 is a view illustrating a tube having a cylindrical dialysis membrane.
  • FIG. 11 is a graph illustrating results of absorbance reduction of the porous silica particles over time.
  • FIG. 12 is a graph and a table illustrating results of absorbance reduction by particle diameter of the porous silica particles over time.
  • FIG. 13 is a graph and a table illustrating results of absorbance reduction by pore diameter of the porous silica particles over time.
  • FIG. 14 is a graph illustrating results of absorbance reduction of the porous silica particles by pH in environments over time.
  • FIG. 15 is a graph illustrating results of absorbance reduction of the porous silica particles over time.
  • FIG. 16 is a view illustrating a tube for identifying release of bioactive material from the porous silica particles.
  • FIG. 17 is a graph illustrating release of bioactive material loaded on the porous silica particles over time.
  • FIG. 18 is micrographs demonstrating siRNA release in mice by loading the porous silica particles with siRNA.
    •  DETAILED DESCRIPTION
  • The terms used in the present invention are defined as follows.
  • “siRNA” refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing. siRNA can inhibit expression of a target gene and may be provided as an efficient gene knockdown method or as a gene therapy method. The siRNA molecule may have a structure in which a sense strand (a sequence corresponding to mRNA sequence of the target gene) and an antisense strand (a complementary sequence to mRNA sequence of the target gene) are located on sides opposite to each other to form a double chain. In addition, siRNA molecules may have a single stranded structure with self-complementary sense and antisense strands. The siRNA is not limited to a complete pair of double-stranded RNA modalities that are paired with each other, but may also include modalities that are not paired due to a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) or the like. The siRNA terminal structure may include blunt or cohesive terminals as long as it can inhibit expression of a target gene by RNA interference (RNAi) effects. The cohesive terminal structure may be a 3′-terminal protruding structure and 5′-terminal protruding structure. Further, siRNA molecules may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and the antisense strands. In this case, the siRNA molecule formed by expression of the nucleotide sequence may form a hairpin structure by intramolecular hybridization, which in turn forms a stem-and-loop structure on the whole. This stem-and-loop structure may be processed in vitro or in vivo to produce siRNA molecules capable of mediating RNAi.
  • “dsRNA” refers to a siRNA precursor molecule that meets a RISC complex containing DICER enzyme (Ribonuclease III) of a target cell and is cleaved into siRNA. In this process, RNAi is generated. dsRNA has a longer sequence by several nucleotides than siRNA and may have a structure wherein a sense strand (a sequence corresponding to mRNA sequence of the target gene) and an antisense strand (a sequence complementary to mRNA sequence of the target gene) are located on sides opposite to each other to form a double chain.
  • “Nucleic acid” may include any DNA or RNA, for example, chromosomes, mitochondria, viruses and/or bacterial nucleic acids present in a tissue sample. One or both strands of a double-stranded nucleic acid molecule may be included, and further any fragment or portion of an intact nucleic acid molecule.
  • “Gene” refers to any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expressions. The gene may include only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may involve gene abnormality in exon, intron, an initiation or termination region, a promoter sequence, another regulatory sequence or a specific sequence adjacent to the gene.
  • As used herein, the term “gene expression” generally refers to a cellular process in which a polypeptide having biological activity is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, the gene expression may include not only transcription and translation processes but also post-transcription and post-translation processes that may affect the biological activity of the gene or gene product. Such processes may include polypeptide synthesis, transport and post-translational modification as well as RNA synthesis, processing and transport, but it is not limited thereto. In the case of a gene which does not encoding a protein product such as siRNA gene, the term “gene expression” refers to a process in which a precursor siRNA is produced from a gene. Normally, the above process is referred to as transcription, although a transcription product of siRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on a protein coding gene. Nevertheless, the formation of mature siRNAs from siRNA genes may be encompassed by the term “gene expression” as that term is used herein.
  • As used herein, the term “target gene” refers to a gene targeted for modulation using the method and composition in the subject matters disclosed herein. Therefore, the target gene includes a nucleic acid sequence with a specific expression level down-regulated by siRNA into mRNA or a polypeptide level. Similarly, the term “target RNA” or “target mRNA” refers to a transcript of the target gene that is bound to siRNA and induces modulation of expression in the target gene.
  • As used herein, the term “transcription” refers to a cellular process involving interaction between an expression-inducible gene, which is RNA of structural information present in a coding sequence of the gene, and RNA polymerase.
  • As used herein, the expression “down-regulation” refers to considerably decreasing the expression of a specific gene into mRNA or the expression level into a protein by gene transcription or gene translation in activated cells, as compared to normal tissue cells.
  • As used herein, the term “treatment” means an approach to obtain beneficial or desired clinical results. For the purposes of the present invention, the beneficial or desired clinical results may include, without limitation, alleviation of symptoms, reduction in an extent of disease, stabilization (i.e., not worsening) of disease state, delayed progression of disease or reduction in progress rate of disease, improvement, temporary mitigation and alleviation of disease state (partially or wholly), whether or not it is detectable. Further, the term “treatment” may also refer to increasing the survival rate compared to that expected survival when untreated. The treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments may include treatments required for disorders that have already occurred as well as disorders to be prevented.
  • As used herein, the term “prevention” means any action to inhibit or delay development of a related disease. It will be apparent to those skilled in the art that the composition mentioned herein may prevent initial symptoms, or related diseases in a case of administering before symptoms appear.
  • Hereinafter, the present invention will be described in detail.
  • The present invention provides a pharmaceutical composition for preventing or treating liver cancer, that is, hepatocellular carcinoma (HCC), which includes: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, as well as antisense RNA having a complementary sequence thereto; or
  • dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.
  • The siRNA or dsRNA of the present invention may be derived from animals including humans, such as monkeys, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, and the like, and is preferably derived from humans.
  • The siRNA or dsRNA of the present invention may be modified by deletion, substitution or insertion of a functional equivalent of nucleic acid molecule constituting the siRNA or dsRNA, for example, a part of the base sequence in the siRNA or dsRNA of the present invention, however, may also be a concept including variants which are capable of functionally performing the same action as the siRNA or dsRNA of the present invention.
  • The siRNA or dsRNA of the present invention may be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or may include commercially available ones. Further, the composition of the present invention may include not only siRNA or dsRNA itself of the present invention but also other substances, for example, compounds, natural products, novel proteins, etc. which are capable of increasing an expression rate of the siRNA or dsRNA of the present invention in cells.
  • Meanwhile, the siRNA or dsRNA of the present invention may be provided in a state of being included in a vector for intracellular expression.
  • The siRNA or dsRNA of the present invention may be introduced into cells by various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and a nuclear protein, a complex of DNA and lipid and the like. To this end, the siRNA or dsRNA of the present invention may be provided in a form of being contained in a carrier enabling efficient introduction into a cell. The carrier is preferably a vector, and both viral vectors and non-viral vectors are usable. As the viral vector may include lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector, etc., preferably, is a lentivirus vector, but it is not limited thereto. Lentivirus is one type of retrovirus characterized by infecting a non-mitotic cell as well as a mitotic cell due to nucleophilic property of pre-integrated complex (a virus “shell”) that allows active incorporation into nucleopores or a complete nuclear membrane.
  • In addition, the vector containing siRNA or dsRNA of the present invention preferably includes a selection marker. The “selection marker” is intended to facilitate selection or screening of cells into which siRNA or dsRNA of the present invention has been introduced. The selection marker used in the vector is not particularly limited as long as it is a gene capable of easily detecting or determining whether or not the vector was introduced. However, examples thereof may typically include markers endowing selectable phenotypes such as drug resistance, auxotrophy, tolerance to cytotoxic agents, expression of surface protein, etc., in particular, green fluorescent protein (GFP), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hisD), guanine phosphoribosyltransferase (Gpt) or the like. Preferably, the green fluorescent protein (GFP) and puromycin markers are used.
  • The composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28 in Table 1 below, and an antisense RNA having a complementary sequence thereto; and dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181 in Table 1 below.
  • In this regard, siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28 in Table 1 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181 in Table 1 below, may target variant 1 sequence (SEQ ID NO: 1) of human BANF1 gene to inhibit expression of the human BANF1 gene variant 1 through RNAi, thereby achieving effects of preventing or treating HCC.
  • TABLE 11
    Target sequence 5: GC content: 45.0%
    5′-CAA GAA GCT GGA SEQ ID NO: 5
    GGA AAG-3′ (SEQ ID Sense strand:
    NO: 482) 5′-CAA GAA GCU GGA GGA AAG
    (Position in UU-3′
    gene sequence: 601) SEQ ID NO: 326
    Antisense strand:
    5′- CUU UCC UCC AGC UUC
    UUG UU-3′
    SEQ ID NO: 158
    dsRNA:
    5′- CAA GAA GCU GGA GGA AAG
    UU UCU AAA G-3′
    Target sequence 6: GC content: 40.9%
    5′-GAA AGA TGA AGA SEQ ID NO: 6
    CCT CTT CC-3′ (SEQ Sense strand:
    ID NO: 483) 5′-GAA AGA UGA AGA CCU CUU
    (Position in CCU U-3′
    gene sequence: 667) SEQ ID NO: 327
    Antisense strand:
    5′-GGA AGA GGU CUU CAU
    CU UCU U-3′
    SEQ ID NO: 159
    dsRNA:
    5′- GAA AGA UGA AGA CCU CUU
    CCU UUC UAA AG-3′
    Target sequence 7: GC content: 40.9%
    5′-GGA ATG GCT GAA SEQ ID NO: 7
    AGA CAC TT-3′ (SEQ Sense strand:
    ID NO: 484) 5′-GGA AUG GCU GAA AGA CAC
    (Position UUU U-3′
    in gene  SEQ ID NO: 328
    sequence: 688) Antisense strand:
    5′-AAG UGU CUU UCA GCC
    AUU CCU U-3′
    SEQ ID NO: 160
    dsRNA:
    5′- GGA AUG GCU GAA AGA CAC
    UUU UUC UAA AG-3′
    Target sequence 8: GC content: 55.0%
    5′-CCA GTG TTC CCA SEQ ID NO: 8
    GTT CCC-3′ (SEQ ID Sense strand:
    NO: 485)  5′-CCA GUG UUC CCA GUU CCC
    (Position in UU-3′
    gene sequence: 1) SEQ ID NO: 329
    Antisense strand:
    5′-GGG AAC UGG GAA CAC
    UGG UU-3′
    SEQ ID NO: 161
    dsRNA:
    5′-CCA GUG UUC CCA GUU CCC
    UU UCU AAA G-3′
    Target sequence 9: GC content: 55.0%
    5′-CCA GTC CAA CTG SEQ ID NO: 9
    CGA GGA-3′ (SEQ ID Sense strand:
    NO: 486) 5′- CCA GUC CAA CUG CGA GGA
    (Position in UU-3′
    gene sequence: 19) SEQ ID NO: 330
    Antisense strand:
    5′-UCCUCGCAGUUGGACUGG UU-3′
    SEQ ID NO: 162
    dsRNA:
    5′- CCAGUCCAACUGCGAGGA UU
    UCU AAA G-3′
    Target sequence 10: GC content: 50.0%
    5′-CGA CGT GAG TCT SEQ ID NO: 10
    GAG TCT-3′ (SEQ ID Sense strand:
    NO: 487)  5′- CGA CGU GAG UCU GAG
    (Position in UCU UU-3′
    gene sequence: 41) SEQ ID NO: 331
    Antisense strand:
    5′-
    AGACUCAGACUCACGUCG UU-3′
    SEQ ID NO: 163
    dsRNA:
    5′- CGACGUGAGUCUGAGUCU UU
    UCU AAA G-3′
    Target sequence 11: GC content: 40.0%
    5′-GTC CGT CTT CTA SEQ ID NO: 11
    ACT CTT-3′ (SEQ ID Sense strand:
    NO: 488)  5′- GUC CGU CUU CUA ACU
    (Position in CUU UU-3′
    gene sequence: 116) SEQ ID NO: 332
    Antisense strand:
    5′- AAGAGUUAGAAGACGGAC
    UU-3′
    SEQ ID NO: 164
    dsRNA:
    5′- GUCCGUCUUCUAACUCUU UU
    UCU AAA G-3′
    Target sequence 12: GC content: 45.0%
    5′-CGT CAA GCC TAA SEQ ID NO: 12
    GTC CTT-3′ (SEQ ID Sense strand:
    NO: 489) 5′- CGUCAAGCCUAAGUCCUU
    (Position in UU-3′
    gene sequence: 149) SEQ ID NO: 333
    Antisense strand:
    5′-
    AAGGACUUAGGCUUGACG UU-3′
    SEQ ID NO: 165
    dsRNA:
    5′- CGUCAAGCCUAAGUCCUU UU
    UCU AAA G-3′
    Target sequence 13: GC content: 45.0%
    5′-GCA GAG AAA GGA SEQ ID NO: 13
    AGT CCT-3′ (SEQ ID Sense strand:
    NO: 490) 5′- GCAGAGAAAGGAAGUCCU
    (Position in UU-3′
    gene sequence: 185) SEQ ID NO: 334
    Antisense strand:
    5′-
    AGGACUUCCUUUCUCUGC UU-3′
    SEQ ID NO: 166 dsRNA:
    5′- GCAGAGAAAGGAAGUCCU UU
    UCU AAA G-3′
    Target sequence 14: GC content: 50.0%
    5′-CGA GAA GCG AGA SEQ ID NO: 14
    CCT TAG-3′ (SEQ ID Sense strand:
    NO: 491) (Position in 5′- CGAGAAGCGAGACCUUAG
    gene sequence: 234) UU-3′
    SEQ ID NO: 335
    Antisense strand:
    5′-CUAAGGUCUCGCUUCUCG UU-3′
    SEQ ID NO: 167
    dsRNA:
    5′- CGAGAAGCGAGACCUUAG UU
    UCU AAA G-3′
    Target sequence 15: GC content: 40.0%
    5′-CCT CAA CTC TAT SEQ ID NO: 15
    AGC TCT-3′ (SEQ ID Sense strand:
    NO: 492) (Position in 5′- CCUCAACUCUAUAGCUCU
    gene sequence: 319) UU-3′
    SEQ ID NO: 336
    Antisense strand:
    5′-AGAGCUAUAGAGUUGAGG
    UU-3′
    SEQ ID NO: 168
    dsRNA:
    5′- CCUCAACUCUAUAGCUCU UU
    UCU AAA G-3′
    Target sequence 16: GC content: 45.0%
    5′-CTA GTG GCT TGA SEQ ID NO: 16
    GGT ATC-3′ (SEQ ID Sense strand:
    NO: 493) 5′- CUAGUGGCUUGAGGUAUC
    (Position in UU-3′
    gene sequence: 423) SEQ ID NO: 337
    Antisense strand:
    5′-
    GAUACCUCAAGCCACUAG UU-3′
    SEQ ID NO: 169
    dsRNA:
    5′- CUAGUGGCUUGAGGUAUC UU
    UCU AAA G-3′
    Target sequence 17: GC content: 40.0%
    5′-GGA TTA AGC CTG SEQ ID NO: 17
    ATC A AG-3′ (SEQ ID Sense strand:
    NO: 494) 5′- GGAUUAAGCCUGAUCAAG
    (Position in UU-3′
    gene sequence: 491) SEQ ID NO. 338
    Antisense strand:
    5′-CUUGAUCAGGCUUAAUCC
    UU-3′
    SEQ ID NO: 170
    dsRNA:
    5′- GGAUUAAGCCUGAUCAAG UU
    UCU AAA G-3′
    Target sequence 18: GC content: 50.0%
    5′-GAC TGC TTC GGA SEQ ID NO: 18
    TGC CTT-3′ (SEQ ID Sense strand:
    NO: 495)  5′- GACUGCUUCGGAUGCCUU
    (Position in UU-3′
    gene sequence: 734) SEQ ID NO: 339
    Antisense strand:
    5′-AAGGCAUCCGAAGCAGUC UU-3′
    SEQ ID NO: 171
    dsRNA:
    5′- GACUGCUUCGGAUGCCUU UU
    UCU AAA G-3′
    Target sequence 19: GC content: 45.0%
    5′-CCT TCT TGT GAT SEQ ID NO: 19
    GCT CTC-3′ (SEQ ID Sense strand:
    NO: 496) 5′- CCUUCUUGUGAUGCUCUC
    (Position in UU-3′
    gene sequence: 768) SEQ ID NO: 340
    Antisense strand:
    5′-
    GAGAGCAUCACAAGAAGG UU-3′
    SEQ ID NO: 172
    dsRNA:
    5′- CCUUCUUGUGAUGCUCUC UU
    UCU AAA G-3′
    Target sequence 20: GC content: 45.0%
    5′-CCT CAT CCA GAG SEQ ID NO: 20 
    TTT GCA-3′ (SEQ ID Sense strand:
    NO: 497) 5′- CCUCAUCCAGAGUUUGCA
    (Position in UU-3′
    gene sequence: 808) SEQ ID NO: 341
    Antisense strand:
    5′-UGCAAACUCUGGAUGAGG UU-3′
    SEQ ID NO: 173
    dsRNA:
    5′- CCUCAUCCAGAGUUUGCA UU
    UCU AAA G-3′
    Target sequence 21: GC content: 50.0%
    5′-CCT GTC CTC TAC SEQ ID NO: 21
    GAA GGA-3′ (SEQ ID Sense strand:
    NO: 498) 5′- CCUGUCCUCUACGAAGGA
    (Position in UU-3′
    gene sequence: 845) SEQ ID NO: 342
    Antisense strand:
    5′-
    UCCUUCGUAGAGGACAGG UU-3′
    SEQ ID NO: 174
    dsRNA:
    5′- CCUGUCCUCUACGAAGGA UU
    UCU AAA G-3′
    Target sequence 22: GC content: 36.36%
    5′-GAT TGC TAT TGT SEQ ID NO: 22 
    CGT ACT CA-3′ Sense strand:
    (SEQ ID NO: 499) 5′-GAUUGCUAUUGUCGUACUCA UU-3′
    (Position in SEQ ID NO: 343
    gene sequence: 866) Antisense strand:
    5′-
    UGAGUACGACAAUAGCAAUC UU-3′
    SEQ ID NO: 175
    dsRNA:
    5′- sense UU UCU AAA G-3′
    Target sequence 23: GC content: 45.0%
    5′-GGA TTC TCG CTC SEQ ID NO: 23
    TTG CAT-3′ (SEQ ID Sense strand:
    NO: 500) 5′- GGAUUCUCGCUCUUGCAU
    (Position in UU-3′
    gene sequence: 947) SEQ ID NO: 344
    Antisense strand:
    5′-AUGCAAGAGCGAGAAUCC UU-3′
    SEQ ID NO: 176
    dsRNA:
    5′- GGAUUCUCGCUCUUGCAU UU
    UCU AAA G-3′
    Target sequence 24: GC content: 45.0%
    5′-GGT GAC AGT TAC SEQ ID NO: 24
    CAG CTT-3′ (SEQ ID Sense strand:
    NO: 501)  5′- GGUGACAGUUACCAGCUU
    (Position in UU-3′
    gene sequence: 999) SEQ ID NO: 345
    Antisense strand:
    5′-AAGCUGGUAACUGUCACC UU-3′
    SEQ ID NO: 177
    dsRNA:
    5′- GGUGACAGUUACCAGCUU UU
    UCU AAA G-3′
    Target sequence 25: GC content: 40.0%
    5′-CCT CAC TTT CAA SEQ ID NO: 25
    TCC GTT-3′ (SEQ ID Sense strand:
    NO: 502)  5′- CCUCACUUUCAAUCCGUU
    (Position in UU-3′
    gene sequence: 1054) SEQ ID NO: 346
    Antisense strand:
    5′-
    AACGGAUUGAAAGUGAGG UU-3′
    SEQ ID NO: 178
    dsRNA:
    5′- CCUCACUUUCAAUCCGUU UU
    UCU AAA G-3′
    Target sequence 26: GC content: 50.0%
    5′-GCA GAA CAG TCA SEQ ID NO: 26
    CTG TCC-3′ (SEQ ID Sense strand:
    NO: 503) 5′- GCAGAACAGUCACUGUCC
    (Position in UU-3′
    gene sequence: 1096) SEQ ID NO: 347
    Antisense strand:
    5′-GGACAGUGACUGUUCUGC UU-3′
    SEQ ID NO: 179
    dsRNA:
    5′- GCAGAACAGUCACUGUCC UU
    UCU AAA G-3′
    Target sequence 27: GC content: 36.36%
    5′-GAT CAA TAA AGT SEQ ID NO: 27
    CAG TGG CT-3′ (SEQ Sense strand:
    ID NO: 504)  5′-GAUCAAUAAAGUCAGUGGCU UU-3′
    (Position in SEQ ID NO: 348
    gene sequence: 1128) Antisense strand:
    5′-AGCCACUGACUUUAUUGAUC UU-3′
    SEQ ID NO: 180
    dsRNA:
    5′- GAUCAAUAAAGUCAGUGGCU
    UU UCU AAA G-3′
    Target sequence 28: GC content: 47.83%
    5′-AAG AAG CTG GAG SEQ ID NO: 28
    GAA AGG GGT -3′ Sense strand:
    (SEQ IDNQ 505) 5′-AAGAAGCUGGAGGAAAGGGGU
    UU-3′
    SEQ ID NO: 349
    Antisense strand:
    5′-CCCCUUUCCUCCGCUUCUU
    UU-3′
    SEQ ID NO: 181
    dsRNA:
    5′- AAGAAGCUGGAGGAAAGGGGU
    UU UCU AAA G-3′
  • The composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55 in Table 2 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208 in Table 2 below.
  • In this regard, siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55 in Table 2 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208 in Table 2 below, may target variant 2 sequence (SEQ ID NO: 2) of human BANF1 gene to inhibit expression of the human BANF1 gene variant 2 through RNAi, thereby achieving effects of preventing or treating HCC.
  • TABLE 2
    Target sequence 29: GC content: 40.9%
    5′-ATG AC A ACC TCC SEQ ID NO: 29
    CAA AAG CA-3′ (SEQ Sense strand:
    ID NO: 506) 5′-AUGACAACCUCCCAAAAGCA
    (Position in UU-3′
    gene sequence: 452)  SEQ ID NO: 350
    Antisense strand:
    5′-UGCUUUUGGGAGGUUGUCAU
    UU-3′
    SEQ ID NO: 182
    dsRNA:
    5′-AUGACAACCUCCCAAAAGCA
    UU UCU AAA G-3′
    Target sequence 30: GC content: 40.9%
    5′-CCG AGA CTT CGT SEQ ID NO: 30
    GGC AGA-3′ (SEQ ID Sense strand:
    NO: 507) 5′- CCGAGACUUCGUGGCAGA
    (Position in UU-3′
    gene sequence: 472) SEQ ID NO: 351
    Antisense strand:
    5′-UCUGCCACGAAGUCUCGG
    UU-3′
    SEQ ID NO: 183
    dsRNA 
    5′- CCGAGACUUCGUGGCAGA UU
    UCU AAA G-3′
    Target sequence 31: GC content: 52.94%
    5′-AGC CTG GCT GGG SEQ ID NO: 31
    ATT-3′ (SEQ ID NO: Sense strand:
    508) (Position 5′- AGCCUGGCUGGGAUU 
    in gene UU-3′
    sequence: 515) SEQ ID NO: 352
    Antisense strand:
    5′- AAUCCCAGCCAGGCU
    UU-3′
    SEQ ID NO: 184
    dsRNA:
    5′- AGCCUGGCUGGGAUU UU
    UCU AAA G-3′
    Target sequence 32:5′- GC content: 42.1%
    CAA GAA GCT GGA SEQ ID NO: 32
    GGA AA-3′ Sense strand:
    (SEQ ID NO: 509) 5′- CAAGAAGCUGGAGGAAA
    (Position UU-3′
    in gene SEQ ID NO: 353
    sequence: 544) Antisense strand:
    5′-
    UUUCCUCCAGCUUCUUG UU-3′
    SEQ ID NO: 185
    dsRNA:
    5′- CAAGAAGCUGGAGGAAA UU
    UCU AAA G-3′
    Target sequence 33: GC content: 40.9%
    5′-CCA GTT TCT GGT SEQ ID NO: 33
    GCT AAA GA-3′ (SEQ Sense strand:
    ID NO: 510)  5′-CCAGUUUCUGGUGCUAAAGA
    (Position in UU-3′
    gene sequence: 592) SEQ ID NO: 354
    Antisense strand:
    5′-UCUUUAGCACCAGAAACUGG
    UU-3′
    SEQ ID NO: 186
    dsRNA:
    5′- CCAGUUUCUGGUGCUAAAGA
    UU UCU AAA G-3′
    Target sequence 34: GC content: 40.0%
    5′-AAG ATG AAG ACC SEQ ID NO: 34
    TCT TCC-3′ (SEQ ID Sense strand:
    NO: 511)  5′- AAGAUGAAGACCUCUUCC
    (Position in UU-3′
    gene sequence: 612) SEQ ID NO: 355
    Antisense strand:
    5′GGAAGAGGUCUUCAUCUU UU-3′
    SEQ ID NO 187
    dsRNA:
    5′- AAGAUGAAGACCUCUUCC UU
    UCU AAA G-3′
    Target sequence 35: GC content: 52.38%
    5′-GGA CTG CTT CGG SEQ ID NO: 35
    ATG CCT T-3′ (SEQ ID Sense strand:5′-
    NO: 512) GGACUGCUUCGGAUGCCUU UU-3′
    (Position in SEQ ID NO: 356
    gene sequence: 676) Antisense strand:
    5′-AAGGCAUCCGAAGCAGUCC
    UU-3′
    SEQ ID NO: 188
    dsRNA:
    5′- GGACUGCUUCGGAUGCCUU
    UU UCU AAA G-3′
    Target sequence 36: GC content: 52.38%
    5′-AGT GGT GCG ACG SEQ ID NO: 36
    CCT TCT T-3′ (SEQ ID Sense strand:
    NO: 513) 5′-AGUGGUGCGACGCCUUCUU
    (Position in UU-3′
    gene sequence: 698) SEQ ID NO: 357
    Antisense strand:
    5′-AAGAAGGCGUCGCACCACU
    UU-3′
    SEQ ID NO: 189
    dsRNA:
    5′- AGUGGUGCGACGCCUUCUU
    UU UU UCU AAA G-3′
    Target sequence 37: GC content: 52.38%
    5′-CTC TCT GGG AAG SEQ ID NO: 37
    CTC TCA AT-3′ Sense strand:
    (SEQ ID NO: 514) 5′-AGUGGUGCGACGCCUUCUU
    (Position in UU-3′
    gene sequence: 724) SEQ ID NO: 358
    Antisense strand:
    5′-AAGAAGGCGUCGCACCACU
    UU-3′
    SEQ ID NO: 190
    dsRNA:
    5′- AGUGGUGCGACGCCUUCUU
    UU UCU AAA G-3′
    Target sequence 38:5′- GC content: 40.9%
    TTG CTA TTG TCG SEQ ID NO: 38
    TAC TCA CC-3′ Sense strand:
    (SEQ ID 5′-UUGCUAUUGUCGUACUCACC
    NO: 515) UU-3′
    (Position in SEQ ID NO: 359
    gene sequence: 811) Antisense strand:
    5′-GGUGAGUACGACAAUAGCAA
    UU-3′
    SEQ ID NO: 191
    dsRNA:
    5′- UUGCUAUUGUCGUACUCACC
    UU UCU AAA G-3′
    Target sequence 39: GC content: 45.0%
    5′-GAT TCT CGC TCT SEQ ID NO: 39
    TGC ATG-3′ (SEQ ID Sense strand:
    NO: 516) 5′- GAUUCUCGCUCUUGCAUG
    (Position in UU-3′
    gene sequence: 891) SEQ ID NO: 360
    Antisense strand:
    5′-CAUGCAAGAGCGAGAAUC UU-3′
    SEQ ID NO: 192
    dsRNA:
    5′- GAUUCUCGCUCUUGCAUG UU
    UCU AAA G-3′
    Target sequence 40: GC content: 45.45%
    5′-CAG TTC CCT GGT SEQ ID NO: 40
    GAC AGT TA-3′ (SEQ Sense strand:
    ID NO: 517) 5′-CAGUUCCCUGGUGACAGUUA
    (Position in UU-3′
    gene sequence: 933) SEQ ID NO: 361
    Antisense strand:5′-
    UAACUGUCACCAGGGAACUG UU-3′
    SEQ ID NO: 193
    dsRNA:
    5′- CAGUUCCCUGGUGACAGUUA
    UU UCU AAA G-3′
    Target sequence 41: GC content: 45.0%
    5′-CCA GCT TTC CTG SEQ ID NO: 41
    AAT GGA-3′ (SEQ ID Sense strand:
    NO: 518) 5′- CCAGCUUUCCUGAAUGGA
    (Position in UU-3′
    gene sequence: 953) SEQ ID NO: 362
    Antisense strand: 5′-
    UCCAUUCAGGAAAGCUGG UU-3′
    SEQ ID NO: 194
    dsRNA:
    5′- CCAGCUUUCCUGAAUGGA UU
    UCU AAA G-3′
    Target sequence 42: GC content: 36.36%
    5′-CTC ACT TTC AAT SEQ ID NO: 42
    CCG TTT GA-3′ (SEQ ID Sense strand:
    NO: 519)  5′-CUCACUUUCAAUCCGUUUGA
    (Position in UU-3′
    gene sequence: 998) SEQ ID NO: 363
    Antisense strand:
    5′-UCAAACGGAUUGAAAGGAG
    UU-3′
    SEQ ID NO: 195
    dsRNA:
    5′- CUCACUUUCAAUCCGUUUGA
    UU UCU AAA G-3′
    Target sequence 43: GC content: 45.45%
    5′-CAG AAC AGT CAC SEQ ID NO: 43
    TGT CCT TG-3′ (SEQ ID Sense strand:
    NO: 520)  5′-CAGAACAGUCACUGUCCUUG
    (Position in UU-3′
    gene sequence: 1039) SEQ ID NO: 364
    Antisense strand:
    5′-CAAGGACAGUGACUGUUCUG
    UU-3′
    SEQ ID NO 196
    dsRNA:
    5′- CAGAACAGUCACUGUCCUUG
    UU UCU AAA G-3′
    Target sequence 44: GC content: 55.0%
    5′-CAC CAG TCC AAC SEQ ID NO: 44
    TGC GAG-3′ (SEQ ID Sense strand:
    NO: 521) 5′- CACCAGUCCAACUGCGAG
    (Position in UU-3′
    gene sequence: 8) SEQ ID NO: 365
    Antisense strand:
    5′-CUCGCAGUUGGACUGGUG
    UU-3′
    SEQ ID NO: 197
    dsRNA:
    5′- CACCAGUCCAACUGCGAG UU
    UCU AAA G-3′
    Target sequence 45:5′- GC content: 50.0%
    TGC GAC GTG AGT SEQ ID NO: 45
    CTG AGT CT-3′ (SEQ ID Sense strand:
    NO: 522) (Position in 5′-UGCGACGUGAGUCUGAGUCU
    gene sequence: 39) UU-3′
    SEQ ID NO: 366
    Antisense strand:
    5′-AGACUCAGACUCACGUCGCA
    UU-3′
    SEQ ID NO: 198
    dsRNA:
    5′- UGCGACGUGAGUCUGAGUCU
    UU UCU AAA G-3′
    Target sequence 46:5′- GC content: 45.0%
    CCT CCG AAA ACC SEQ ID NO: 46
    GTA CTT-3′ (SEQ ID Sense strand:
    NO: 523) (Position in 5′- CCUCCGAAAACCGUACUU
    gene sequence: 63) UU-3′
    SEQ ID NO: 367
    Antisense strand:
    5′-AAGUACGGUUUUCGGAGG
    UU-3′
    SEQ ID NO: 199
    dsRNA:
    5′- CCUCCGAAAACCGUACUU UU
    UCU AAA G-3′
    Target sequence 47: GC content: 45.45%
    5′-CCT TGT CCG TCT TCT SEQ ID NO: 47
    AAC TC-3′ (SEQ ID NO: Sense strand:
    524) 5′-CCUUGUCCGUCUUCUAACUC
    (Position in gene UU-3′
    sequence: 113) SEQ ID NO: 368
    Antisense strand:
    5′-GAGUUAGAAGACGGACAAGG
    UU-3′
    SEQ ID NO: 200
    dsRNA:
    5′- CCUUGUCCGUCUUCUAACUC
    UU UCU AAA G-3′
    Target sequence 48: GC content: 52.38%
    5′-CCA GGT CCG TCA SEQ ID NO: 48
    AGC CTA A-3′ (SEQ ID Sense strand:
    NO: 525) 5′-CCAGGUCCGUCAAGCCUAA
    (Position in UU-3′
    gene sequence: 142) SEQ ID NO. 369
    Antisense strand:
    5′-UUAGGCUUGACGGACCUGG
    UU-3′
    SEQ ID NO: 201
    dsRNA:
    5′- CCAGGUCCGUCAAGCCUAA
    UU UCU AAA G-3′
    Target sequence 49: GC content: 45.0%
    5′-GCA GCA GAG AAA SEQ ID NO 49
    GGA AGT-3′ (SEQ ID Sense strand:
    NO: 526)  5′- GCAGCAGAGAAAGGAAGU
    (Position in UU-3′
    gene sequence: 182) SEQ ID NO: 370
    Antisense strand:
    5′-UACUUCCUUUCUCUGCUGC
    UU-3′
    SEQ ID NO: 202
    dsRNA:
    5′- GCAGCAGAGAAAGGAAGU UU
    UCU AAA G-3′
    Target sequence 50: GC content: 45.0%
    5′-CCT ATC TCC CTC SEQ ID NO: 50
    AGA ACT-3′ (SEQ ID Sense strand:
    NO: 527) 5′- CCUAUCUCCCUCAGAACU
    (Position in UU-3′
    gene sequence: 214) SEQ ID NO: 371
    Antisense strand:
    5′-AGUUCUGAGGGAGAUAGG
    UU-3′
    SEQ ID NO: 203
    dsRNA:
    5′- CCUAUCUCCCUCAGAACU UU
    UCU AAA G-3′
    Target sequence 51: GC content: 45.45%
    5′-GAG AAG CGA GAC SEQ ID NO: 51
    CTT AGA AG-3′ (SEQ Sense strand:
    ID NO: 528) 5′-GAGAAGCGAGACCUUAGAAG
    (Position in UU-3′
    gene sequence: 236) SEQ ID NO: 372
    Antisense strand:
    5′-CUUCUAAGGUCUCGCUUCUC
    UU-3′
    SEQ ID NO: 204
    dsRNA:
    5′- GAGAAGCGAGACCUUAGAAG
    UU UCU AAA G-3′
    Target sequence 52: GC content: 40.9%
    5′-GCC TCA ACT CTA SEQ ID NO: 52
    TAG CTC TA-3′ (SEQ ID Sense strand:
    NO: 529)  5′-GCCUCAACUCUAUAGCUCUA
    (Position in UU-3′
    gene sequence: 319) SEQ ID NO: 373
    Antisense strand:
    5′-UAGAGCUAUAGAGUUGAGGC
    UU-3′
    SEQ ID NO: 205
    dsRNA:
    5′- GCCUCAACUCUAUAGCUCUA
    UU UCU AAA G-3′
    Target sequence 53: GC content: 40.0%
    5′-CCA ACG TGG AAT SEQ ID NO: 53
    GTT TCT-3′ (SEQ ID Sense strand:
    NO: 530) 5′- CCAACGUGGAAUGUUUCU
    (Position in UU-3′
    gene sequence: 354) SEQ ID NO: 374
    Antisense strand:
    5′-AGAAACAUUCCACGUUGG UU-3′
    SEQ ID NO 206
    dsRNA:
    5′- CCAACGUGGAAUGUUUCU UU
    UCU AAA G-3′
    Target sequence 54: GC content: 41.67%
    5′-GAA GCG GAA GTG SEQ ID NO: 54
    GAA GAA AGT T-3′ Sense strand:
    (SEQ ID NO: 531) 5′-GAAGCGGAAGUGGAAGAAAGUU
    (Position in gene UU-3′
    sequence: 401) SEQ ID NO: 375
    Antisense strand:
    5′-AACUUUCUUCCACUUCCGCUUC
    UU-3′
    SEQ ID NO: 207
    dsRNA:
    5′-GAAGCGGAAGUGGAAGAAAGUU
    UU UCU AAA G-3′
    Target sequence 55: GC content: 40.9%
    5′-CTA GTG GCT TGA SEQ ID NO: 55
    GAT TAA GC-3′ (SEQ Sense strand:
    ID NO: 532) 5′-CUAGUGGCUUGAGAUUAAGC
    (Position in UU-3′
    gene sequence: 423) SEQ ID NO: 376
    Antisense strand:
    5′-GCUUAAUCUCAAGCCACUAG
    UU-3′
    SEQ ID NO: 208
    dsRNA:
    5′- CUAGUGGCUUGAGAUUAAGC
    UU UCU AAA G-3′
  • The composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120 in Table 3 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273 in Table 3 below.
  • In this regard, siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120 in Table 3 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273 in Table 3 below, may target a sequence of human PLOD3 gene (SEQ ID NO: 3) to inhibit expression of the human PLOD3 gene through RNAi, thereby achieving effects of preventing or treating HCC.
  • TABLE 3
    Target sequence 56: 5′- GC content: 50.0%
    CCA GAG AAG CTG SEQ ID NO: 56 Sense strand: 5′-
    CTG GTG AT-3′ (SEQ CCAGAGAAGCUGCUGGUGAU UU-3′
    ID NO: 533) (Position in SEQ ID NO: 377 Antisense strand: 5′-
    gene sequence: 562) AUCACCAGCAGCUUCUCUGG UU-3′
    SEQ ID NO: 209 dsRNA: 5′-CCAGAGAAGCUGCUGGUGAU
    UU UCU AAA G-3′
    Target sequence 57: 5′- GC content: 55.0%
    CCA CAG CTG AAA SEQ ID NO: 57 Sense strand: 5′-CCACAGCUGAAACCGAGG
    CCG AGG-3′ (SEQ ID UU-3′
    NO: 534) (Position in SEQ ID NO: 378 Antisense strand: 5′-
    gene sequence: 590) CCUCGGUUUCAGCUGUGG UU-3′
    SEQ ID NO: 210 dsRNA: 5′-CCACAGCUGAAACCGAGG UU
    UCU AAA G-3′
    Target sequence 58: 5′- GC content: 50.0%
    CTC TGC GGA GTT SEQ ID NO: 58 Sense strand: 5′-CUCUGCGGAGUUCUUC UU-
    CTT C-3′ (SEQ ID NO: 3′
    535) (Position in gene SEQ ID NO: 379 Antisense strand: 5′-GAAGAACUCCGCAGAG
    sequence: 627) UU-3′
    SEQ ID NO: 211 dsRNA: 5′-CUCUGCGGAGUUCUUC UU
    UCU AAA G-3′
    Target sequence 59: 5′- GC content: 50.0%
    AAC TAC ACT GTG SEQ ID NO: 59 Sense strand: 5′-AACUACACUGUGCGGACC
    CGG ACC-3′ (SEQ ID UU-3′
    NO: 536) (Position in SEQ ID NO: 380 Antisense strand: 5′-
    gene sequence: 643) GGUCCGCACAGUGUAGUU UU-3′
    SEQ ID NO: 212 dsRNA: 5′-AACUACACUGUGCGGACC UU
    UCU AAA G-3′
    Target sequence 60: 5′- GC content: 50.0%
    GTG ATG TGG CTC SEQ ID NO: 60 Sense strand: 5′-GUGAUGUGGCUCGAACAG
    GAA CAG-3′ (SEQ ID UU-3′
    NO: 537) (Position in SEQ ID NO: 381 Antisense strand:5′-
    gene sequence: 689) CUGUUCGAGCCACAUCAC UU-3′
    SEQ ID NO: 213 dsRNA: 5′-GUGAUGUGGCUCGAACAG UU
    UCU AAA G-3′
    Target sequence 61: 5′- GC content: 36.36%
    GGT TAA AGA AGG SEQ ID NO: 61 Sense strand: 5′-
    AAA TGG AG-3′ (SEQ GGUUAAAGAAGGAAAUGGAG UU-3′
    ID NO: 538) (Position in SEQ ID NO: 382 Antisense strand: 5′-
    gene sequence: 731) CUCCAUUUCCUUCUUUAACC UU-3′
    SEQ ID NO: 214 dsRNA: 5′-GGUUAAAGAAGGAAAUGGAG
    UU UCU AAA G-3′
    Target sequence 62: 5′- GC content: 36.36%
    GGA GGA TAT GAT SEQ ID NO: 62 Sense strand: 5′-
    CAT CAT GT-3′ (SEQ GGAGGAUAUGAUCAUCAUGU UU-3′
    ID NO: 539) (Position in SEQ ID NO: 383 Antisense strand: 5′-
    gene sequence: 765) ACAUGAUGAUCAUAUCCUCC UU-3′
    SEQ ID NO: 215 dsRNA: 5′-GGAGGAUAUGAUCAUCAUGU
    UU UCU AAA G-3′
    Target sequence 63: 5′- GC content: 45.0%
    GGA TAG CTA CGA SEQ ID NO: 63 Sense strand: 5′-GGAUAGCUACGACGUGAU
    CGT GAT-3′ (SEQ ID UU-3′
    NO: 540) (Position in SEQ ID NO: 384 Antisense strand: 5′-
    gene sequence: 789) AUCACGUCGUAGCUAUCC UU-3′
    SEQ ID NO: 216 dsRNA: 5′-GGAUAGCUACGACGUGAU UU
    UCU AAA G-3′
    Target sequence 64: 5′- GC content: 45.0%
    CAC AGA GCT GCT SEQ ID NO: 64 Sense strand: 5′-CACAGAGCUGCUGAAGAA
    GAA GAA-3′ (SEQ ID UU-3′
    NO: 541) (Position in SEQ ID NO: 385 Antisense strand: 5′-
    gene sequence: 822) UUCUUCAGCAGCUCUGUG UU-3′
    SEQ ID NO: 217 dsRNA: 5′-CACAGAGCUGCUGAAGAA UU
    UCU AAA G-3′
    Target sequence 65: 5′- GC content: 45.0%
    TGC TCT TCT CTG SEQ ID NO: 65 Sense strand: 5′-UGCUCUUCUCUGCAGAGA
    CAG AGA-3′ (SEQ ID UU-3′
    NO: 542) (Position in SEQ ID NO: 386 Antisense strand: 5′-
    gene sequence: 863) UCUCUGCAGAGAAGAGCA UU-3′
    SEQ ID NO: 218 dsRNA: 5′-UGCUCUUCUCUGCAGAGA UU
    UCU AAA G-3′
    Target sequence 66: 5′- GC content: 47.62%
    GCT TCC TCA ATT SEQ ID NO: 66 Sense strand: 5′-GCUUCCUCAAUUCUGGUGG
    CTG GTG G-3′ (SEQ ID UU-3′
    NO: 543) (Position in SEQ ID NO: 387 Antisense strand: 5′-
    gene sequence: 941) CCACCAGAAUUGAGGAAGC UU-3′
    SEQ ID NO: 219 dsRNA: 5′-GCUUCCUCAAUUCUGGUGG UU
    UCU AAA G-3′
    Target sequence 67: 5′- GC content: 40.9%
    ATT CAT CGG TTT SEQ ID NO: 67 Sense strand: 5′-
    TGC CAC CA-3′ (SEQ AUUCAUCGGUUUUGCCACCA UU-3′
    ID NO: 544) (Position in SEQ ID NO: 388 Antisense strand: 5′-
    gene sequence: 950) UGGUGGCAAAACCGAUGAAU UU-3′
    SEQ ID NO: 220 dsRNA: 5′-AUUCAUCGGUUUUGCCACCA
    UU UCU AAA G-3′
    Target sequence 68: 5′- GC content: 38.1%
    AGT GGA AGT ACA SEQ ID NO: 68 Sense strand: 5′-AGUGGAAGUACAAGGAUGA
    AGG ATG A-3′ (SEQ UU-3′
    ID NO: 545) (Position in SEQ ID NO: 389 Antisense strand: 5′-
    gene sequence: 1001) UCAUCCUUGUACUUCCACU UU-3′
    SEQ ID NO: 221 dsRNA: 5′-AGUGGAAGUACAAGGAUGA
    UU UCU AAA G-3′
    Target sequence 69: 5′- GC content: 40.0%
    CAG CCT TAA TCT SEQ ID NO: 69 Sense strand: 5′-CAGCCUUAAUCUGGAUCA
    GGA TCA-3′ (SEQ ID UU-3′
    NO: 546) (Position in SEQ ID NO: 390 Antisense strand: 5′-
    gene sequence: 1080) UGAUCCAGAUUAAGGCUG UU-3′
    SEQ ID NO: 222 dsRNA: 5′-CAGCCUUAAUCUGGAUCA UU
    UCU AAA G-3′
    Target sequence 70: 5′- GC content: 45.45%
    GTC TCG GAT CTT SEQ ID NO: 70 Sense strand: 5′-
    TCA GAA CC-3′ (SEQ GUCUCGGAUCUUUCAGAACC UU-3′
    ID NO: 547) (Position in SEQ ID NO: 391 Antisense strand: 5′-
    gene sequence: 1101) GGUUCUGAAAGAUCCGAGAC UU-3′
    SEQ ID NO: 223 dsRNA: 5′-GUCUCGGAUCUUUCAGAACC
    UU UCU AAA G-3′
    Target sequence 71: 5′- GC content: 40.0%
    GGC TTT AGA TGA SEQ ID NO: 71 Sense strand: 5′-GGCUUUAGAUGAAGUGGU
    AGT GGT-3′ (SEQ ID UU-3′
    NO: 548) (Position in SEQ ID NO: 392 Antisense strand: 5′-
    gene sequence: 1128) ACCACUUCAUCUAAAGCC UU-3′
    SEQ ID NO: 224 dsRNA: 5′-GGCUUUAGAUGAAGUGGU UU
    UCU AAA G-3′
    Target sequence 72: 5′- GC content: 45.0%
    GTT TGA TCG GAA SEQ ID NO: 72 Sense strand: 5′-GUUUGAUCGGAACCGUGU
    CCG TGT-3′ (SEQ ID UU-3′
    NO: 549) (Position in SEQ ID NO: 393 Antisense strand: 5′-
    gene sequence: 1152) ACACGGUUCCGAUCAAAC UU-3′
    SEQ ID NO: 225 dsRNA: 5′-GUUUGAUCGGAACCGUGU UU
    UCU AAA G-3′
    Target sequence 73: 5′- GC content: 45.0%
    TTG TGG TCC ATG SEQ ID NO: 73 Sense strand: 5′-UUGUGGUCCAUGGAAACG
    GAA ACG-3′ (SEQ ID UU-3′
    NO: 550) (Position in SEQ ID NO: 394 Antisense strand: 5′-
    gene sequence: 1205) CGUUUCCAUGGACCACAA UU-3′
    SEQ ID NO: 226 dsRNA: 5′-UUGUGGUCCAUGGAAACG UU
    UCU AAA G-3′
    Target sequence 74: 5′- GC content: 47.62%
    CCA CTA AGC TGC SEQ ID NO: 74 Sense strand: 5′-CCACUAAGCUGCAGCUCAA
    AGC TCA A-3′ (SEQ ID UU-3′
    NO: 551) (Position in SEQ ID NO: 395 Antisense strand: 5′-
    gene sequence: 1223) UUGAGCUGCAGCUUAGUGG UU-3′
    SEQ ID NO: 227 dsRNA: 5′-CCACUAAGCUGCAGCUCAA UU
    UCU AAA G-3′
    Target sequence 75: 5′- GC content: 52.38%
    CCA ATG GCT GGA SEQ ID NO: 75 Sense strand: 5′-CCAAUGGCUGGACUCCUGA
    CTC CTG A-3′ (SEQ ID UU-3′
    NO: 552) (Position in SEQ ID NO: 396 Antisense strand: 5′-
    gene sequence: 1265) TCAGGAGTCCAGCCATTGG UU-3′
    SEQ ID NO: 228 dsRNA: 5′-CCAAUGGCUGGACUCCUGA UU
    UCU AAA G-3′
    Target sequence 76: 5′- GC content: 52.38%
    GCT GTG GCT TCT SEQ ID NO: 76 Sense strand: 5′-GCUGUGGCUUCUGCAACCA
    GCA ACC A-3′ (SEQ ID UU-3′
    NO: 553) (Position in SEQ ID NO: 397 Antisense strand: 5′-
    gene sequence: 1289) UGGUUGCAGAAGCCACAGC UU-3′
    SEQ ID NO: 229 dsRNA: 5′-GCUGUGGCUUCUGCAACCA UU
    UCU AAA G-3′
    Target sequence 77: 5′- GC content: 45.0%
    GTG TTT GTG GAA SEQ ID NO: 77 Sense strand: 5′-GUGUUUGUGGAACAGCCU
    CAG CCT-3′ (SEQ ID UU-3′
    NO: 554) (Position in SEQ ID NO: 398 Antisense strand: 5′-
    gene sequence: 1360) AGGCUGUUCCACAAACAC UU-3′
    SEQ ID NO: 230 dsRNA: 5′-GUGUUUGUGGAACAGCCU UU
    UCU AAA G-3′
    Target sequence 78: 5′- GC content: 47.62%
    GCT GCT ACT CCT SEQ ID NO: 78 Sense strand: 5′-GCUGCUACUCCUGGACUAU
    GGA CTA T-3′ (SEQ ID UU-3′
    NO: 555) (Position in SEQ ID NO: 399 Antisense strand: 5′-
    gene sequence: 1407) AUAGUCCAGGAGUAGCAGC UU-3′
    SEQ ID NO: 231 dsRNA: 5′-GCUGCUACUCCUGGACUAU UU
    UCU AAA G-3′
    Target sequence 79: 5′- GC content: 45.45%
    TTC CTG CAC AAC SEQ ID NO: 79 Sense strand: 5′-
    AAC GAG GT-3′ (SEQ UUCCUGCACAACAACGAGGU UU-3′
    ID NO: 556) (Position in SEQ ID NO: 400 Antisense strand: 5′-
    gene sequence: 1447) ACCUCGUUGUUGUGCAGGAA UU-3′
    SEQ ID NO: 232 dsRNA: 5′-UUCCUGCACAACAACGAGGU
    UU UCU AAA G-3′
    Target sequence 80: 5′- GC content: 55.0%
    CCA CAT CGC TGA SEQ ID NO: 80 Sense strand: 5′-CCACAUCGCUGACUCCUG
    CTC CTG-3′ (SEQ ID UU-3′
    NO: 557) (Position in SEQ ID NO: 401 Antisense strand: 5′-
    gene sequence: 1479) CAGGAGUCAGCGAUGUGG UU-3′
    SEQ ID NO: 233 dsRNA: 5′-CCACAUCGCUGACUCCUG UU
    UCU AAA G-3′
    Target sequence 81: 5′- GC content: 50.0%
    AGC TCC AGG ACC SEQ ID NO: 81 Sense strand: 5′-
    ACT TCT CA-3′ (SEQ AGCUCCAGGACCACUUCUCA UU-3′
    ID NO: 558) (Position in SEQ ID NO: 402 Antisense strand: 5′-
    gene sequence: 1502) TGAGAAGTGGTCCTGGAGCT UU-3′
    SEQ ID NO: 234 dsRNA: 5′-AGCUCCAGGACCACUUCUCA
    UU UCU AAA G-3′
    Target sequence 82: 5′- GC content: 50.0%
    ATG GCC ATG GAC SEQ ID NO: 82 Sense strand: 5′-AUGGCCAUGGACCUGUGU
    CTG TGT-3′ (SEQ ID UU-3′
    NO: 559) (Position in SEQ ID NO: 403 Antisense strand: 5′-
    gene sequence: 1576) ACACAGGUCCAUGGCCAU UU-3′
    SEQ ID NO: 235 dsRNA: 5′-AUGGCCAUGGACCUGUGU UU
    UCU AAA G-3′
    Target sequence 83: 5′- GC content: 40.9%
    CGA GTG TGA GTT SEQ ID NO: 83 Sense strand: 5′-
    CTA CTT CA-3′ (SEQ CGAGUGUGAGUUCUACUUCA UU-3′
    ID NO: 560) (Position in SEQ ID NO: 404 Antisense strand:5′-
    gene sequence: 1605) UGAAGUAGAACUCACACUCG UU-3′
    SEQ ID NO: 236 dsRNA: 5′-CGAGUGUGAGUUCUACUUCA
    UU UCU AAA G-3′
    Target sequence 84: 5′- GC content: 55.0%
    GCT GTC CTC ACC SEQ ID NO: 84 Sense strand: 5′-GCUGUCCUCACCAACCUG
    AAC CTG-3′ (SEQ ID UU-3′
    NO: 561) (Position in SEQ ID NO: 405 Antisense strand: 5′-
    gene sequence: 1639) CAGGUUGGUGAGGACAGC UU-3′
    SEQ ID NO: 237 dsRNA: 5′-GCUGUCCUCACCAACCUG UU
    UCU AAA G-3′
    Target sequence 85: 5′- GC content: 45.0%
    CTG CGT ATC CTC SEQ ID NO: 85 Sense strand: 5′-CUGCGUAUCCUCAUUGAG
    ATT GAG-3′ (SEQ ID UU-3′
    NO: 562) (Position in SEQ ID NO: 406 Antisense strand: 5′-
    gene sequence: 1663) CUCAAUGAGGAUACGCAG UU-3′
    SEQ ID NO: 238 dsRNA: 5′-CUGCGUAUCCUCAUUGAG UU
    UCU AAA G-3′
    Target sequence 86: 5′- GC content: 45.0%
    GAG AAC AGG AAG SEQ ID NO: 86 Sense strand: 5′-GAGAACAGGAAGGUGAUC
    GTG ATC-3′ (SEQ ID UU-3′
    NO: 563) (Position in SEQ ID NO: 407 Antisense strand: 5′-
    gene sequence: 1681) GAUCACCUUCCUGUUCUC UU-3′
    SEQ ID NO: 239 dsRNA: 5′-GAGAACAGGAAGGUGAUC UU
    UCU AAA G-3′
    Target sequence 87: 5′- GC content: 45.0%
    CAA GCT GTG GTC SEQ ID NO: 87 Sense strand: 5′-CAAGCUGUGGUCCAACUU
    CAA CTT-3′ (SEQ ID UU-3′
    NO: 564) (Position in SEQ ID NO: 408 Antisense strand: 5′-
    gene sequence: 1722) AAGUUGGACCACAGCUUG UU-3′
    SEQ ID NO: 240 dsRNA: 5′-CAAGCUGUGGUCCAACUU UU
    UCU AAA G-3′
    Target sequence 88: 5′- GC content: 55.0%
    GAG GAC TAC GTG SEQ ID NO: 88 Sense strand: 5′-GAGGACUACGUGGAGCUG
    GAG CTG-3′ (SEQ ID UU-3′
    NO: 565) (Position in SEQ ID NO: 409 Antisense strand: 5′-
    gene sequence: 1780) CAGCUCCACGUAGUCCUC UU-3′
    SEQ ID NO: 241 dsRNA: 5′-GAGGACUACGUGGAGCUG UU
    UCU AAA G-3′
    Target sequence 89: 5′- GC content: 40.9%
    GTG TGT GGA ATG SEQ ID NO: 89 Sense strand: 5′-
    TAC CAT AC-3′ (SEQ GUGUGUGGAAUGUACCAUAC UU-3′
    ID NO: 566) (Position in SEQ ID NO: 410 Antisense strand: 5′-
    gene sequence: 1817) GUAUGGUACAUUCCACACAC UU-3′
    SEQ ID NO: 242 dsRNA: 5′-GUGUGUGGAAUGUACCAUAC
    UU UCU AAA G-3′
    Target sequence 90: 5′- GC content: 52.38%
    AGA GGG ATG TGT SEQ ID NO: 90 Sense strand: 5′-AGAGGGAUGUGUUCUCGGG
    TCT CGG G-3′ (SEQ ID UU-3′
    NO: 567) (Position in SEQ ID NO: 411 Antisense strand: 5′-
    gene sequence: 1888) CCCGAGAACACAUCCCUCU UU-3′
    SEQ ID NO: 243 dsRNA: 5′-AGAGGGAUGUGUUCUCGGG
    UU UCU AAA G-3′
    Target sequence 91: 5′- GC content: 40.9%
    CCT TCT GTA AGA SEQ ID NO: 91 Sense strand: 5′-
    GCT TTC GA-3′ (SEQ CCUUCUGUAAGAGCUUUCGA UU-3′
    ID NO: 568) (Position in SEQ ID NO: 412 Antisense strand: 5′-
    gene sequence: 1931) UCGAAAGCUCUUACAGAAGG UU-3′
    SEQ ID NO: 244 dsRNA: 5′-CCUUCUGUAAGAGCUUUCGA
    UU UCU AAA G-3′
    Target sequence 92: 5′- GC content: 45.45%
    ACA AGG GCA TCT SEQ ID NO: 92 Sense strand: 5′-
    TCC TCC AT-3′ (SEQ ACAAGGGCAUCUUCCUCCAU UU-3′
    ID NO: 569) (Position in SEQ ID NO: 413 Antisense strand: 5′-
    gene sequence: 1952) AUGGAGGAAGAUGCCCUUGU UU-3′
    SEQ ID NO: 245 dsRNA: 5′-ACAAGGGCAUCUUCCUCCAU
    UU UCU AAA G-3′
    Target sequence 93: 5′- GC content: 40.0%
    CTG AGC AAT CAG SEQ ID NO: 93 Sense strand: 5′-CUGAGCAAUCAGCAUGAA
    CAT GAA-3′ (SEQ ID UU-3′
    NO: 570) (Position in SEQ ID NO: 414 Antisense strand: 5′-
    gene sequence: 1972) UUCAUGCUGAUUGCUCAG UU-3′
    SEQ ID NO: 246 dsRNA: 5′-CUGAGCAAUCAGCAUGAAUU
    UCU AAA G-3′
    Target sequence 94: 5′- GC content: 45.0%
    CCA CTT CCA GAT SEQ ID NO: 94 Sense strand: 5′-CCACUUCCAGAUACGACA
    ACG ACA-3′ (SEQ ID UU-3′
    NO: 571) (Position in SEQ ID NO: 415 Antisense strand: 5′-
    gene sequence: 2006) UGUCGUAUCUGGAAGUGG UU-3′
    SEQ ID NO: 247 dsRNA: 5′-CCACUUCCAGAUACGACA UU
    UCU AAA G-3′
    Target sequence 95: 5′- GC content: 47.62%
    ACC TCT GGC AGA SEQ ID NO: 95 Sense strand: 5′-ACCUCUGGCAGAUCUUCGA
    TCT TCG A-3′ (SEQ ID UU-3′
    NO: 572) (Position in SEQ ID NO: 416 Antisense strand: 5′-
    gene sequence: 2042) UCGAAGAUCUGCCAGAGGU UU-3′
    SEQ ID NO: 248 dsRNA: 5′-ACCUCUGGCAGAUCUUCGA UU
    UCU AAA G-3′
    Target sequence 96: 5′- GC content: 55.0%
    CGT CGA CTG GAA SEQ ID NO: 96 Sense strand: 5′-CGUCGACUGGAAGGAGCA
    GGA GCA-3′ (SEQ ID UU-3′
    NO: 573) (Position in SEQ ID NO: 417 Antisense strand: 5′-
    gene sequence: 2067) UGCUCCUUCCAGUCGACG UU-3′
    SEQ ID NO: 249 dsRNA: 5′-CGUCGACUGGAAGGAGCA UU
    UCU AAA G-3′
    Target sequence 97: 5′- GC content: 40.0%
    GTA CAT CCA CGA SEQ ID NO: 97 Sense strand: 5′-GUACAUCCACGAGAACUA
    GAA CTA-3′ (SEQ ID UU-3′
    NO: 574) (Position in SEQ ID NO: 418 Antisense strand: 5′-
    gene sequence: 2085) UAGUUCUCGUGGAUGUAC UU-3′
    SEQ ID NO: 250 dsRNA: 5′-GUACAUCCACGAGAACUA UU
    UCU AAA G-3′
    Target sequence 985′- GC content: 50.0%
    AAG GAA TCG TGG SEQ ID NO: 98 Sense strand: 5′-
    AGC AGC CA-3′ (SEQ AAGGAAUCGUGGAGCAGCCA UU-3′
    ID NO: 575) (Position in SEQ ID NO: 419 Antisense strand: 5′-
    gene sequence: 2123) UGGCUGCUCCACGAUUCCUU UU-3′
    SEQ ID NO: 251 dsRNA: 5′-AAGGAAUCGUGGAGCAGCCA
    UU UCU AAA G-3′
    Target sequence 995′- GC content: 40.0%
    CTG CTG TCA GAA SEQ ID NO: 99 Sense strand: 5′-CUGCUGUCAGAACAAAUG
    CAA ATG-3′ (SEQ ID UU-3′
    NO: 576) (Position in SEQ ID NO: 420 Antisense strand: 5′-
    gene sequence: 2167) CAUUUGUUCUGACAGCAG UU-3′
    SEQ ID NO: 252 dsRNA: 5′-CUGCUGUCAGAACAAAUG UU
    UCU AAA G-3′
    Target sequence 100: 5′- GC content: 50.0%
    TGT GAT GAG CTG SEQ ID NO: 100 Sense strand: 5′-
    GTG GCA GA-3′ (SEQ UGUGAUGAGCUGGUGGCAGA UU-3′
    ID NO: 577) (Position in SEQ ID NO: 421 Antisense strand: 5′-
    gene sequence: 2185) UCUGCCACCAGCUCAUCACA UU-3′
    SEQ ID NO: 253 dsRNA: 5′-UGUGAUGAGCUGGUGGCAGA
    UU UCU AAA G-3′
    Target sequence 101: 5′- GC content: 45.0%
    GCA TGA GGA TTC SEQ ID NO: 101 Sense strand: 5′-GCAUGAGGAUUCAAGGCU
    AAG GCT-3′ (SEQ ID UU-3′
    NO: 578) (Position in SEQ ID NO: 422 Antisense strand: 5′-
    gene sequence: 2238) AGCCUUGAAUCCUCAUGC UU-3′
    SEQ ID NO: 254 dsRNA: 5′-GCAUGAGGAUUCAAGGCU UU
    UCU AAA G-3′
    Target sequence 102: 5′- GC content: 47.62%
    CTG GAG GCT ACG SEQ ID NO: 102 Sense strand: 5′-
    AGA ATG T-3′ (SEQ ID CUGGAGGCUACGAGAAUGU UU-3′
    NO: 579) (Position in SEQ ID NO: 423 Antisense strand: 5′-
    gene sequence: 2256) ACAUUCUCGUAGCCUCCAG UU-3′
    SEQ ID NO: 255 dsRNA: 5′-CUGGAGGCUACGAGAAUGU UU
    UCU AAA G-3′
    Target sequence 103: 5′- GC content: 45.0%
    TGG ACA TCC ACA SEQ ID NO: 103 Sense strand: 5′-UGGACAUCCACAUGAAGC
    TGA AGC-3′ (SEQ ID UU-3′
    NO: 580) (Position in SEQ ID NO: 424 Antisense strand: 5′-
    gene sequence: 2285) GCUUCAUGUGGAUGUCCA UU-3′
    SEQ ID NO: 256 dsRNA: 5′-UGGACAUCCACAUGAAGC UU
    UCU AAA G-3′
    Target sequence 104: 5′- GC content: 54.54%
    TAC GAG GAC CAG SEQ ID NO: 104 Sense strand: 5′-
    TGG CTG CA-3′ (SEQ UACGAGGACCAGUGGCUGCA UU-3′
    ID NO: 581) (Position in SEQ ID NO: 425 Antisense strand: 5′-
    gene sequence: 2311) TGCAGCCACTGGTCCTCGTA UU-3′
    SEQ ID NO: 257 dsRNA: 5′-UACGAGGACCAGUGGCUGCA
    UU UCU AAA G-3′
    Target sequence 105: 5′- GC content: 47.62%
    CAT GAC CGA GAG SEQ ID NO: 105 Sense strand: 5′-
    CCT GTT T-3′ (SEQ ID CAUGACCGAGAGCCUGUUU UU-3′
    NO: 582) (Position in SEQ ID NO: 426 Antisense strand:5′-
    gene sequence: 2355) AAACAGGCUCUCGGUCAUG UU-3′
    SEQ ID NO: 258 dsRNA: 5′-CAUGACCGAGAGCCUGUUU UU
    UCU AAA G-3′
    Target sequence 106: 5′- GC content: 40.9%
    GTG ATG AAC TTT SEQ ID NO: 106 Sense strand: 5′-
    GTG GTT CG-3′ 3′ GUGAUGAACUUUGUGGUUCG UU-3′
    (SEQ ID NO: 583) SEQ ID NO: 427 Antisense strand: 5′-
    (Position in gene CGAACCACAAAGUUCAUCAC UU-3′
    sequence: 2401) SEQ ID NO: 259 dsRNA: 5′-GUGAUGAACUUUGUGGUUCG
    UU UCU AAA G-3′
    Target sequence 107: 5′- GC content: 55.0%
    AGA CGA GCA GCC SEQ ID NO: 107 Sense strand: 5′-AGACGAGCAGCCGUCUCU
    GTC TCT-3′ (SEQ ID UU-3′
    NO: 584) (Position in SEQ ID NO: 428 Antisense strand: 5′-
    gene sequence: 2429) AGAGACGGCUGCUCGUCU UU-3′
    SEQ ID NO: 260 dsRNA: 5′-AGACGAGCAGCCGUCUCU UU
    UCU AAA G-3′
    Target sequence 108: 5′- GC content: 50.0%
    GAC TCA TCC ACC SEQ ID NO: 108 Sense strand: 5′-
    TTC ACC CT-3′ (SEQ GACUCAUCCACCUUCACCCU UU-3′
    ID NO: 585) (Position in SEQ ID NO: 429 Antisense strand: 5′-
    gene sequence: 2461) AGGGUGAAGGUGGAUGAGUC UU-3′
    SEQ ID NO: 261 dsRNA: 5′-
    GACUCAUCCACCUUCACCCUUU UCU AAA G-3′
    Target sequence 109: 5′- GC content: 50.0%
    TTC CTG CGC TAC SEQ ID NO: 109 Sense strand: 5′-
    GAC TGT GT-3′ (SEQ UUCCUGCGCUACGACUGUGU UU-3′
    ID NO: 586) (Position in SEQ ID NO: 430 Antisense strand: 5′-
    gene sequence: 2533) ACACAGUCGUAGCGCAGGAA UU-3′
    SEQ ID NO: 262 dsRNA: 5′-UUCCUGCGCUACGACUGUGU
    UU UCU AAA G-3′
    Target sequence 110: 5′- GC content: 45.45%
    CAC ACG CTA CAT SEQ ID NO: 110 Sense strand: 5′-
    CAT GGT GT-3′ (SEQ CACACGCUACAUCAUGGUGU UU-3′
    ID NO: 587) (Position in SEQ ID NO: 431 Antisense strand: 5′-
    gene sequence: 2637) ACACCAUGAUGUAGCGUGUG UU-3′
    SEQ ID NO: 263 dsRNA: 5′-CACACGCUACAUCAUGGUGU
    UU UCU AAA G-3′
    Target sequence 111: 5′- GC content: 40.9%
    TGC CAT TGT GCC SEQ ID NO: 111 Sense strand: 5′-
    TTT TTA GG-3′ (SEQ UGCCAUUGUGCCUUUUUAGG UU-3′
    ID NO: 588) (Position in SEQ ID NO: 432 Antisense strand: 5′-
    gene sequence: 2701) CCUAAAAAGGCACAAUGGCA UU-3′
    SEQ ID NO: 264 dsRNA: 5′-UGCCAUUGUGCCUUUUUAGG
    UU UCU AAA G-3′
    Target sequence 112: 5′- GC content: 40.9%
    CAC TTC CTG AGT SEQ ID NO: 112 Sense strand: 5′-
    TCA TGT TC-3′ (SEQ CACUUCCUGAGUUCAUGUUC UU-3′
    ID NO: 589) (Position in SEQ ID NO: 433 Antisense strand: 5′-
    gene sequence: 2769) GAACAUGAACUCAGGAAGUG UU-3′
    SEQ ID NO: 265 dsRNA: 5′-CACUUCCUGAGUUCAUGUUC
    UU UCU AAA G-3′
    Target sequence 113: 5′- GC content: 40.9%
    CCT GAA CTG AAT SEQ ID NO: 113 Sense strand: 5′-
    ATG TCA CC-3′ (SEQ CCUGAACUGAAUAUGUCACC UU-3′
    ID NO: 590) (Position in SEQ ID NO: 434 Antisense strand: 5′-
    gene sequence: 2796) GGUGACAUAUUCAGUUCAGG UU-3′
    SEQ ID NO: 266 dsRNA: 5′-CCUGAACUGAAUAUGUCACC
    UU UCU AAA G-3′
    Target sequence 114: 5′- GC content: 40.9%
    CGC AGT CTC ACT SEQ ID NO: 114 Sense strand: 5′-
    CTG AAT AAA-3′ CGCAGUCUCACUCUGAAUAAA UU-3′
    (SEQ ID NO: 591) SEQ ID NO: 435 Antisense strand: 5′-
    (Position in gene UUUAUUCAGAGUGAGACUGCG UU-3′
    sequence: 2937) SEQ ID NO: 267 dsRNA: 5′-
    CGCAGUCUCACUCUGAAUAAAUU UCU AAA G-3′
    Target sequence 115: 5′- GC content: 38.06%
    GGA CAG TTT GTA SEQ ID NO: 115 Sense strand: 5′-
    AGT CTT G-3′ (SEQ ID GGACAGUUUGUAAGUCUUG UU-3′
    NO: 592) (Position in SEQ ID NO: 436 Antisense strand: 5′-
    gene sequence: 2958) CAAGACUUACAAACUGUCC UU-3′
    SEQ ID NO: 268 dsRNA: 5′-GGACAGUUUGUAAGUCUUG
    UU UCU AAA G-3′
    Target sequence 116: 5′- GC content: 50.0%
    TCA CTT CCC CTG SEQ ID NO: 116 Sense strand: 5′-
    TCC AGG TT-3′ (SEQ UCACUUCCCCUGUCCAGGUU UU-3′
    ID NO: 593) (Position in SEQ ID NO: 437 Antisense strand: 5′-
    gene sequence: 121) AACCUGGACAGGGGAAGUGA UU-3′
    SEQ ID NO: 269 dsRNA: 5′-UCACUUCCCCUGUCCAGGUU
    UU UCU AAA G-3′
    Target sequence 117: 5′- GC content: 40.9%
    TCA GCT TCC ACA SEQ ID NO: 117 Sense strand: 5′-
    TGT GTC AA-3′ (SEQ UCAGCUUCCACAUGUGUCAA UU-3′
    ID NO: 594) (Position in SEQ ID NO: 438 Antisense strand: 5′-
    gene sequence: 141) UUGACACAUGUGGAAGCUGA UU-3′
    SEQ ID NO: 270 dsRNA: 5′-UCAGCUUCCACAUGUGUCAA
    UU UCU AAA G-3′
    Target sequence 118: 5′- GC content: 47.62%
    GAC AAT CCT CGC SEQ ID NO: 118 Sense strand: 5′-
    CTT GTC T-3′ (SEQ ID GACAAUCCUCGCCUUGUCU UU-3′
    NO: 595) (Position in SEQ ID NO: 439 Antisense strand: 5′-
    gene sequence: 241) AGACAAGGCGAGGAUUGUC UU-3′
    SEQ ID NO: 271 dsRNA: 5′-GACAAUCCUCGCCUUGUCU UU
    UCU AAA G-3′
    Target sequence 119: 5′- GC content: 45.45%
    CAT CTG GAG CTT SEQ ID NO: 119 Sense strand: 5′-
    TCT GTA GC-3′ (SEQ GCAUCUGGAGCUUUCUGUA UU-3′
    ID NO: 596) (Position in SEQ ID NO: 440 Antisense strand: 5′-
    gene sequence: 270) UACAGAAAGCUCCAGAUGC UU-3′
    SEQ ID NO: 272 dsRNA: 5′-GCAUCUGGAGCUUUCUGUA UU
    UCU AAA G-3′
    Target sequence 120: 5′- GC content: 55.0%
    GAG ATC CCA GGA SEQ ID NO: 120 Sense strand: 5′-GAGAUCCCAGGAUCCUGG
    TCC TGG-3′ (SEQ ID UU-3′
    NO: 597) (Position in SEQ ID NO: 441 Antisense strand: 5′-
    gene sequence: 414) CCAGGAUCCUGGGAUCUC UU-3′
    SEQ ID NO: 273 dsRNA: 5′-GAGAUCCCAGGAUCCUGG UU
    UCU AAA G-3′
  • The composition of the present invention may include: siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157 in Table 4 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310 in Table 4 below.
  • In this regard, siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157 in Table 4 below, and an antisense RNA having a complementary sequence thereto; or dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310 in Table 4 below, may target a sequence of human SF3B4 gene (SEQ ID NO: 4) to inhibit expression of the human SF3B4 gene through RNAi, thereby achieving effects of preventing or treating HCC.
  • TABLE 4
    Target sequence 121: 5′- GC content: 40.9%
    AAT CAG GAT GCC SEQ ID NO: 121 Sense strand: 5′-
    ACT GTG TA-3′ (SEQ ID AAUCAGGAUGCCACUGUGUA UU-3′
    NO: 598) (Position in SEQ ID NO: 442 Antisense strand: 5′-
    gene sequence: 521) UACACAGUGGCAUCCUGAUU UU-3′
    SEQ ID NO: 274 dsRNA: 5′-AAUCAGGAUGCCACUGUGUA
    UU UCU AAA G-3′
    Target sequence 122: 5′- GC content: 40.9%
    CTG GAT GAG AAG SEQ ID NO: 122 Sense strand: 5′-
    GTT AGT GA-3′ (SEQ ID CUGGAUGAGAAGGUUAGUGA UU-3′
    NO: 599) (Position in SEQ ID NO: 443 Antisense strand: 5′-
    gene sequence: 551) UCACUAACCUUCUCAUCCAG UU-3′
    SEQ ID NO: 275 dsRNA: 5′-CUGGAUGAGAAGGUUAGUGA
    UU UCU AAA G-3′
    Target sequence 123: 5′- GC content: 45.45%
    TGT GGG AAC TGT SEQ ID NO: 123 Sense strand: 5′-
    TTC TCC AG-3′ (SEQ ID UGUGGGAACUGUUUCUCCAG UU-3′
    NO: 600) (Position in SEQ ID NO: 444 Antisense strand: 5′-
    gene sequence: 579) CUGGAGAAACAGUUCCCACA UU-3′
    SEQ ID NO: 276 dsRNA: 5′-UGUGGGAACUGUUUCUCCAG
    UU UCU AAA G-3′
    Target sequence 124: 5′- GC content: 45.0%
    CTG GAC CAG TAG SEQ ID NO: 124 Sense strand: 5′-
    TCA ACA-3′ (SEQ ID CUGGACCAGUAGUCAACA UU-3′
    NO: 601) (Position in SEQ ID NO: 445 Antisense strand: 5′-
    gene sequence: 599) UGUUGACUACUGGUCCAG UU-3′
    SEQ ID NO: 277 dsRNA: 5′-CUGGACCAGUAGUCAACA UU
    UCU AAA G-3′
    Target sequence 125: 5′- GC content: 42.86%
    CCA AAG GAT AGA SEQ ID NO: 125 Sense strand: 5′-
    GTC ACT G-3′ (SEQ ID CCAAAGGAUAGAGUCACUG UU-3′
    NO: 602) (Position in SEQ ID NO: 446 Antisense strand: 5′-
    gene sequence: 626) CAGUGACUCUAUCCUUUGG UU-3′
    SEQ ID NO: 278 dsRNA: 5′-CCAAAGGAUAGAGUCACUG
    UU UCU AAA G-3′
    Target sequence 126: 5′- GC content: 47.83%
    CAG CAC CAA GGC SEQ ID NO: 126 Sense strand: 5′-
    TAT GGC TTT-3′ (SEQ CAGCACCAAGGCUAUGGCUUU UU-3′
    ID NO: 603) (Position in SEQ ID NO: 447 Antisense strand: 5′-
    gene sequence: 647) AAAGCCAUAGCCUUGGUGCUG UU-3′
    SEQ ID NO: 279 dsRNA: 5′-
    CAGCACCAAGGCUAUGGCUUU UU UCU AAA G-3′
    Target sequence 127: 5′- GC content: 40.9%
    GTG GAA TTC TTG SEQ ID NO: 127 Sense strand: 5′-
    AGT GAG GA-3′ (SEQ GUGGAAUUCUUGAGUGAGGA UU-3′
    ID NO: 604) (Position in SEQ ID NO: 448 Antisense strand: 5′-
    gene sequence: 668) UCCUCACUCAAGAAUUCCAC UU-3′
    SEQ ID NO: 280 dsRNA: 5′-GUGGAAUUCUUGAGUGAGGA
    UU UCU AAA G-3′
    Target sequence 128: 5′- GC content: 36.36%
    GCT GAC TAT GCC SEQ ID NO: 128 Sense strand: 5′-
    ATT AAG AT-3′ (SEQ ID GCUGACUAUGCCAUUAAGAU UU-3′
    NO: 605) (Position in SEQ ID NO: 449 Antisense strand: 5′-
    gene sequence: 692) AUCUUAAUGGCAUAGUCAGC UU-3′
    SEQ ID NO: 281 dsRNA: 5′-GCUGACUAUGCCAUUAAGAU
    UU UCU AAA G-3′
    Target sequence 129: 5′- GC content: 33.33%
    ACA TGA TCA AAC SEQ ID NO: 129 Sense strand: 5′-
    TCT ATG G-3′ (SEQ ID ACAUGAUCAAACUCUAUGG UU-3′
    NO: 606) (Position in SEQ ID NO: 450 Antisense strand: 5′-
    gene sequence: 717) CCAUAGAGUUUGAUCAUGU UU-3′
    SEQ ID NO: 282 dsRNA: 5′-ACAUGAUCAAACUCUAUGG
    UU UCU AAA G-3′
    Target sequence 130: 5′- GC content: 45.0%
    GGT GAA CAA AGC SEQ ID NO: 130 Sense strand: 5′-
    ATC AGC-3′ (SEQ ID GGUGAACAAAGCAUCAGC UU-3′
    NO: 607) (Position in SEQ ID NO: 451 Antisense strand: 5′-
    gene sequence: 748) GCUGAUGCUUUGUUCACC UU-3′
    SEQ ID NO: 283 dsRNA: 5′-GGUGAACAAAGCAUCAGC UU
    UCU AAA G-3′
    Target sequence 131: 5′- GC content: 40.0%
    CCT GAG ATT GAT SEQ ID NO: 131 Sense strand: 5′-
    GAG AAG-3′ (SEQ ID CCUGAGAUUGAUGAGAAG UU-3′
    NO: 608) (Position in SEQ ID NO: 452 Antisense strand: 5′-
    gene sequence: 818) CUUCUCAUCAAUCUCAGG UU-3′
    SEQ ID NO: 284 dsRNA: 5′-CCUGAGAUUGAUGAGAAG UU
    UCU AAA G-3′
    Target sequence 132: 5′- GC content: 42.1%
    GGT CAT CTT ACA SEQ ID NO: 132 Sense strand: 5′-GGUCAUCUUACAAACCC
    AAC CC-3′ (SEQ ID NO: UU-3′
    609) (Position in gene SEQ ID NO: 453 Antisense strand: 5′-
    sequence: 865) GGGUUUGUAAGAUGACC UU-3′
    SEQ ID NO: 285 dsRNA: 5′-GGUCAUCUUACAAACCC UU
    UCU AAA G-3′
    Target sequence 133: 5′- GC content: 55.0%
    CCT GAC ACA GGC SEQ ID NO: 133 Sense strand: 5′-CCUGACACAGGCAACUCC
    AAC TCC-3′ (SEQ ID UU-3′
    NO: 610) (Position in SEQ ID NO: 454 Antisense strand: 5′-
    gene sequence: 899) GGAGUUGCCUGUGUCAGG UU-3′
    SEQ ID NO: 286 dsRNA: 5′-CCUGACACAGGCAACUCC UU
    UCU AAA G-3′
    Target sequence 134: 5′- GC content: 40.9%
    GCT TCA TTT GAT SEQ ID NO: 134 Sense strand: 5′-
    GCT TCG GA-3′ (SEQ ID GCUUCAUUUGAUGCUUCGGA UU-3′
    NO: 611) (Position in SEQ ID NO: 455 Antisense strand: 5′-
    gene sequence: 941) UCCGAAGCAUCAAAUGAAGC UU-3′
    SEQ ID NO: 287 dsRNA: 5′-GCUUCAUUUGAUGCUUCGGA
    UU UCU AAA G-3′
    Target sequence 135: 5′- GC content: 40.9%
    TGC AGC AAT TGA SEQ ID NO: 135 Sense strand: 5′-
    AGC CAT GA-3′ (SEQ UGCAGCAAUUGAAGCCAUGA UU-3′
    ID NO: 612) (Position in SEQ ID NO: 456 Antisense strand: 5′-
    gene sequence: 961) UCAUGGCUUCAAUUGCUGCA UU-3′
    SEQ ID NO: 288 dsRNA: 5′-UGCAGCAAUUGAAGCCAUGA
    UU UCU AAA G-3′
    Target sequence 136: 5′- GC content: 47.62%
    GCA GTA CCT CTG SEQ ID NO: 136 Sense strand: 5′-
    TAA CCG T-3′ (SEQ ID GCAGUACCUCUGUAACCGU UU-3′
    NO: 613) (Position in SEQ ID NO: 457 Antisense strand: 5′-
    gene sequence: 985) ACGGUUACAGAGGUACUGC UU-3′
    SEQ ID NO: 289 dsRNA: 5′-GCAGUACCUCUGUAACCGU
    UU UCU AAA G-3′
    Target sequence 137: 5′- GC content: 40.9%
    CAC CGT ATC TTA SEQ ID NO: 137 Sense strand: 5′-
    TGC CTT CA-3′ (SEQ ID CACCGUAUCUUAUGCCUUCA UU-3′
    NO: 614) (Position in SEQ ID NO: 458 Antisense strand: 5′-
    gene sequence: 1009) UGAAGGCAUAAGAUACGGUG UU-3′
    SEQ ID NO: 290 dsRNA: 5′-CACCGUAUCUUAUGCCUUCA
    UU UCU AAA G-3′
    Target sequence 138: 5′- GC content: 50.0%
    GAA CGA CTT CTG SEQ ID NO: 138 Sense strand: 5′-
    GCA GCT CA-3′ (SEQ GAACGACUUCUGGCAGCUCA UU-3′
    ID NO: 615) (Position in SEQ ID NO: 459 Antisense strand: 5′-
    gene sequence: 1067) UGAGCUGCCAGAAGUCGUUC UU-3′
    SEQ ID NO: 291 dsRNA: 5′-GAACGACUUCUGGCAGCUCA
    UU UCU AAA G-3′
    Target sequence 139: 5′- GC content: 45.45%
    CCT CAT CAG CTG SEQ ID NO: 139 Sense strand: 5′-
    TTT GCA GA-3′ (SEQ ID CCUCAUCAGCUGUUUGCAGA UU-3′
    NO: 616) (Position in SEQ ID NO: 460 Antisense strand: 5′-
    gene sequence: 1112) UCUGCAAACAGCUGAUGAGG UU-3′
    SEQ ID NO: 292 dsRNA: 5′-CCUCAUCAGCUGUUUGCAGA
    UU UCU AAA G-3′
    Target sequence 140: 5′- GC content: 45.45%
    TGG TCA TGG ACA SEQ ID NO: 140 Sense strand: 5′-
    CTC ACA TC-3′ (SEQ ID UGGUCAUGGACACUCACAUC UU-3′
    NO: 617) (Position in SEQ ID NO: 461 Antisense strand: 5′-
    gene sequence: 1351) GAUGUGAGUGUCCAUGACCA UU-3′
    SEQ ID NO: 293 dsRNA: 5′-UGGUCAUGGACACUCACAUC
    UU UCU AAA G-3′
    Target sequence 141: 5′- GC content: 45.0%
    GAT GTC TCA GAT SEQ ID NO: 141 Sense strand: 5′-
    GCA GCT-3′ (SEQ ID GAUGUCUCAGAUGCAGCU UU-3′
    NO: 618) (Position in SEQ ID NO: 462 Antisense strand: 5′-
    gene sequence: 1408) AGCUGCAUCUGAGACAUC UU-3′
    SEQ ID NO: 294 dsRNA: 5′-GAUGUCUCAGAUGCAGCU UU
    UCU AAA G-3′
    Target sequence 142: 5′- GC content: 45.0%
    CCT CAT GGC TTA SEQ ID NO: 142 Sense strand: 5′-
    GGA CAT-3′ (SEQ ID CCUCAUGGCUUAGGACAU UU-3′
    NO: 619) (Position in SEQ ID NO: 463 Antisense strand: 5′-
    gene sequence: 1439) AUGUCCUAAGCCAUGAGG UU-3′
    SEQ ID NO: 295 dsRNA: 5′-CCUCAUGGCUUAGGACAU UU
    UCU AAA G-3′
    Target sequence 143: 5′- GC content: 40.9%
    TCA CAT TTT CCT TCC SEQ ID NO: 143 Sense strand: 5′-
    TCC TG-3′ (SEQ ID NO: UCACAUUUUCCUUCCUCCUG UU-3′
    620) (Position in gene SEQ ID NO: 464 Antisense strand: 5′-
    sequence: 1771) CAGGAGGAAGGAAAAUGUGA UU-3′
    SEQ ID NO: 296 dsRNA: 5′-UCACAUUUUCCUUCCUCCUG
    UU UCU AAA G-3′
    Target sequence 144: 5′- GC content: 45.45%
    CCT TGG ACC AAT SEQ ID NO: 144 Sense strand: 5′-
    CAG AGA TG-3′ (SEQ CCUUGGACCAAUCAGAGAUG UU-3′
    ID NO: 621) (Position in SEQ ID NO: 465 Antisense strand: 5′-
    gene sequence: 1818) CAUCUCUGAUUGGUCCAAGG UU-3′
    SEQ ID NO: 297 dsRNA: 5′-CCUUGGACCAAUCAGAGAUG
    UU UCU AAA G-3′
    Target sequence 145: 5′- GC content: 40.9%
    GGC AAA GGT ACT SEQ ID NO: 145 Sense strand: 5′-
    AAT CCC TT-3′ (SEQ ID GGCAAAGGUACUAAUCCCUU UU-3′
    NO: 622) (Position in SEQ ID NO: 466 Antisense strand: 5′-
    gene sequence: 1852) AAGGGAUUAGUACCUUUGCC UU-3′
    SEQ ID NO: 298 dsRNA: 5′-GGCAAAGGUACUAAUCCCUU
    UU UCU AAA G-3′
    Target sequence 146: 5′- GC content: 40.0%
    TTC CAC AGG AGG SEQ ID NO: 146 Sense strand: 5′-
    TAT TTC-3′ (SEQ ID UUCCACAGGAGGUAUUUC UU-3′
    NO: 623) (Position in SEQ ID NO: 467 Antisense strand: 5′-
    gene sequence: 1911) GAAAUACCUCCUGUGGAA UU-3′
    SEQ ID NO: 299 dsRNA: 5′-UUCCACAGGAGGUAUUUC UU
    UCU AAA G-3′
    Target sequence 147: 5′- GC content: 40.0%
    GGT CCT GAG TAT SEQ ID NO: 147 Sense strand: 5′-
    TTT GCA-3′ (SEQ ID GGUCCUGAGUAUUUUGCA UU-3′
    NO: 624) (Position in SEQ ID NO: 468 Antisense strand: 5′-
    gene sequence: 1940) UGCAAAAUACUCAGGACC UU-3′
    SEQ ID NO: 300 dsRNA: 5′-GGUCCUGAGUAUUUUGCA UU
    UCU AAA G-3′
    Target sequence 148: 5′- GC content: 42.86%
    CCA AAT CTG CAA SEQ ID NO: 148 Sense strand: 5′-
    GAA GGC T-3′ (SEQ ID CCAAAUCUGCAAGAAGGCU UU-3′
    NO: 625) (Position in SEQ ID NO: 469 Antisense strand: 5′-
    gene sequence: 18) AGCCUUCUUGCAGAUUUGG UU-3′
    SEQ ID NO: 301 dsRNA: 5′-CCAAAUCUGCAAGAAGGCU
    UU UCU AAA G-3′
    Target sequence 149: 5′- GC content: 43.48%
    GGA ACT CTT CAG SEQ ID NO: 149 Sense strand: 5′-
    CAC ATC CTT-3′ (SEQ GGAACUCUUCAGCACAUCCUU UU-3′
    ID NO: 626) (Position in SEQ ID NO: 470 Antisense strand: 5′-
    gene sequence: 95) AAGGAUGUGCUGAAGAGUUCC UU-3′
    SEQ ID NO: 302 dsRNA: 5′-
    GGAACUCUUCAGCACAUCCUU UU UCU AAA G-3′
    Target sequence 150: 5′- GC content: 40.9%
    CTC TGG ACA ACA SEQ ID NO: 150 Sense strand: 5′-
    GAA GAA GA-3′ (SEQ CUCUGGACAACAGAAGAAGA UU-3′
    ID NO: 627) (Position in SEQ ID NO: 471 Antisense strand: 5′-
    gene sequence: 116) UCUUCUUCUGUUGUCCAGAG UU-3′
    SEQ ID NO: 303 dsRNA: 5′-CUCUGGACAACAGAAGAAGA
    UU UCU AAA G-3′
    Target sequence 151: 5′- GC content: 40.0%
    TGA GAG CAG TGT SEQ ID NO: 151 Sense strand: 5′-
    GAT TCT-3′ (SEQ ID UGAGAGCAGUGUGAUUCU UU-3′
    NO: 628) (Position in SEQ ID NO: 472 Antisense strand: 5′-
    gene sequence: 201) AGAAUCACACUGCUCUCA UU-3′
    SEQ ID NO: 304 dsRNA: 5′-UGAGAGCAGUGUGAUUCU UU
    UCU AAA G-3′
    Target sequence 152: 5′- GC content: 42.1%
    CAA GTC TAG CAG SEQ ID NO: 152 Sense strand: 5′-CAAGUCUAGCAGUGCAU
    TGC AT-3′ (SEQ ID NO: UU-3′
    629) (Position in gene SEQ ID NO: 473 Antisense strand: 5′-
    sequence: 221) AUGCACUGCUAGACUUG UU-3′
    SEQ ID NO: 305 dsRNA: 5′-CAAGUCUAGCAGUGCAU UU
    UCU AAA G-3′
    Target sequence 153: 5′- GC content: 42.86%
    CTC GCT AAG ACA SEQ ID NO: 153 Sense strand: 5′-
    ACT AGC A-3′ (SEQ ID CUCGCUAAGACAACUAGCA UU-3′
    NO: 630) (Position in SEQ ID NO: 474 Antisense strand: 5′-
    gene sequence: 270) UGCUAGUUGUCUUAGCGAGA UU-3′
    SEQ ID NO: 306 dsRNA: 5′-CUCGCUAAGACAACUAGCA
    UU UCU AAA G-3′
    Target sequence 154: 5′- GC content: 45.45%
    CAG GTT AAG TTT SEQ ID NO: 154 Sense strand: 5′-
    CGG AGG CT-3′ (SEQ CAGGUUAAGUUUCGGAGGCU UU-3′
    ID NO: 631) (Position in SEQ ID NO: 475 Antisense strand: 5′-
    gene sequence: 331) AGCCUCCGAAACUUAACCUG UU-3′
    SEQ ID NO: 307 dsRNA: 5′-CAGGUUAAGUUUCGGAGGCU
    UU UCU AAA G-3′
    Target sequence 155: 5′- GC content: 54.54%
    GCT TCC AGG CAC SEQ ID NO: 155 Sense strand: 5′-
    CTC CTC TT-3′ (SEQ ID GCUUCCAGGCACCUCCUCUU UU-3′
    NO: 632) (Position in SEQ ID NO: 476 Antisense strand: 5′-
    gene sequence: 369) AAGAGGAGGUGCCUGGAAGC UU-3′
    SEQ ID NO: 308 dsRNA: 5′-GCUUCCAGGCACCUCCUCUU
    UU UCU AAA G-3′
    Target sequence 156: 5′- GC content: 50.0%
    GAA GTG GAA GTC SEQ ID NO: 156 Sense strand: 5′-
    GTG CTG AG-3′ (SEQ GAAGUGGAAGUCGUGCUGAG UU-3′
    ID NO: 633) (Position in SEQ ID NO: 477 Antisense strand: 5′-
    gene sequence: 427) CUCAGCACGACUUCCACUUC UU-3′
    SEQ ID NO: 309 dsRNA: 5′-GAAGUGGAAGUCGUGCUGAG
    UU UCU AAA G-3′
    Target sequence 157: 5′- GC content: 50.0%
    GAT CTC TTT CGC SEQ ID NO: 157 Sense strand: 5′-
    CAT GGC TG-3′ (SEQ ID GAUCUCUUUCGCCAUGGCUG UU-3′
    NO: 634) (Position in SEQ ID NO: 478 Antisense strand: 5′-
    gene sequence: 481) CAGCCAUGGCGAAAGAGAUC UU-3′
    SEQ ID NO: 310 dsRNA: 5′-GAUCUCUUUCGCCAUGGCUG
    UU UCU AAA G-3′
  • The siRNA or dsRNA of the present invention may be one capable of being loaded on a carrier while carrying RNA molecules depending on types of the carrier, which is not particularly limited as long as it is known in the art, and may include, but is not limited to, at least one selected from the group consisting of, for example, liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin, lipid particles, polyethylene glycol, poly-L-guluronic alginate, polyglycolic-polylactic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and hydrogel, preferably, porous silica particles having advantages such as high RNA retention, sustained release, biodegradability, etc.
  • The siRNA or dsRNA of the present invention may be loaded on porous silica particles, wherein the particles are particles of silica material (SiO2) and have a nano-sized particle diameter.
  • The porous silica particles may be porous particles having nano-sized pores and may carry physiologically active substances (“bioactive materials”) such as siRNA or dsRNA of the present invention on the surfaces of the particles and/or insides of the pores.
  • The porous silica particles are biodegradable particles and, when the particles loaded with the bioactive material and is administered in a body, may release the bioactive material while being biodegraded in the body. That is, biodegradation of the porous silica particles results in release of the bioactive material. In this case, the porous silica particles according to the present invention may be slowly degraded in the body so that the loaded bioactive material can have sustained release properties.
  • For example, t when a ratio of absorbance in the following Equation 1 becomes ½ may be 20 or more.

  • At/A0   [Equation 1]
  • (wherein A0 is absorbance of the porous silica particles measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particles into a cylindrical dialysis membrane having pores with a diameter of 50 kDa,
  • 15 ml of the same solvent as the suspension is placed outside the dialysis membrane while being in contact with the dialysis membrane, followed by horizontal agitation at 60 rpm and 37° C. inside and outside the dialysis membrane,
  • pH of the suspension is 7.4, and
  • At is absorbance of the porous silica particles measured after t hours elapses from the measurement of A0).
  • Equation 1 indicates how fast the porous silica particles are degraded under environments similar to the body.
  • In Equation 1, the absorbance Ao and At may be measured, for example, after placing the porous silica particles and the suspension in a cylindrical dialysis membrane, and further placing the same suspension on the outside of the dialysis membrane.
  • The particles are biodegradable and may be slowly degraded in the suspension, wherein the diameter of 50 kDa corresponds to about 5 nm, the biodegraded particles can pass through a 50 kDa dialysis membrane, this cylindrical dialysis membrane is under horizontal agitation at 60 rpm such that the suspension is evenly admixed, and the degraded particles may come out of the dialysis membrane.
  • The absorbance in Equation 1 may be measured, for example, under an environment in which the suspension outside the dialysis membrane is replaced with a new suspension. The suspension may be one that is constantly replaced, one that is replaced at a constant period wherein the constant period may be periodic or irregular. For example, the replacement may be performed within a range of 1 hour to 1 week, in particular, at 1-, 2-, 3-, 6-, 12-, 24-hours intervals, or 2-, 3-, 4-, 7-days interval, etc., 2 0 but it is not limited thereto.
  • A ratio of absorbance of ½ means that, after t hours, the absorbance becomes half of the initial absorbance, therefore, means that approximately half of the porous silica particles have been degraded.
  • The suspension may be a buffer solution and, for example, at least one selected from the group consisting of phosphate buffered saline (PBS) and simulated body fluid (SBF), and more specifically, PBS.
  • t when the ratio of absorbance in Equation 1 becomes ½ is 20 or more or 24 or more, for example, t may be 20 to 120, specifically, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65, etc. within the above range, but it is not limited thereto.
  • The particles are characterized in that t when the ratio of absorbance in Equation 1 becomes ⅕ may be, for example, 70 to 140, specifically, 80 to 140, 80 to 120, 80 to 110, 70 to 140, 70 to 120, 70 to 110, etc. within the above range, but it is not limited thereto.
  • The particles are characterized in that t when the ratio of absorbance in Equation 1 becomes 1/20 may be, for example, 130 to 220, specifically, 130 to 200, 140 to 200, 140 to 180, 150 to 180, etc. within the above range, but it is not limited thereto.
  • The particles are characterized in that t when the measured absorbance becomes 0.01 or less may be, for example, 250 or more, specifically, 300 or more, 350 or more, 400 or more, 500 or more, 1000 or more, etc. within the above range while having an upper limit of 2000, but it is not limited thereto.
  • The particles are characterized in that the absorbance ratio in Equation 1 has high positive correlation with t, specifically, Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more, 0.95 or more, etc.
  • t in Equation 1 means how fast the porous silica particles are degraded under environments similar to the body, for example, may be controlled by adjusting the surface area, particle diameter, pore diameter, substituent on the surface of the porous silica particle and/or inside the pore, compactness of the surface, etc.
  • More particularly, t may be reduced by increasing the surface area of the particle or may be increased by reducing the surface area thereof. The surface area may be regulated by adjusting the diameter of the particles and/or the diameter of the pores. In addition, placing a substituent on the surface of the particle and/or the inside of the pore may reduce direct exposure of the porous silica particles to the environment (such as a solvent), thereby increasing t. Further, loading the bioactive material on the porous silica particles and increasing affinity between the bioactive material and the porous silica particles may reduce direct exposure of the porous silica particles to the environment, thereby increasing t. In addition, the surface may be made more densely in the preparation of the particles so as to increase t. In the above, various examples of adjusting tin Equation 1 have been described, but it is not limited thereto.
  • The porous silica particles may be, for example, spherical particles, but it is not limited thereto.
  • The average diameter of the porous silica particles may be, for example, 100 to 1000 nm, specifically, 100 to 800 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, 100 to 200 nm, etc. within the above range, but it is not limited thereto.
  • The average pore diameter of the particles may be, for example, 1 to 100 nm, specifically, 4 to 100 nm, 4 to 50 nm, 4 to 30 nm, 10 to 30 nm, etc. within the above range, but it is not limited thereto. Due to the large pore diameter, the particles may carry a large amount of the bioactive material and/or the bioactive material having a large size.
  • The porous silica particles may have a BET surface area of, for example, 200 to 700 m2/g, specifically, 200 to 700 m2/g, 200 to 650 m2/g, 250 to 650 m2/g, 300 to 700 m2/g, 300 to 650 m2/g, 300 to 600 m2/g, 300 to 550 m2/g, 300 to 500 m2/g, 300 to 450 m2/g, etc. within the above range, but it is not limited thereto.
  • The porous silica particles may have a volume per gram (g) of, for example, 0.7 to 2.2 ml, specifically, 0.7 to 2.0 ml, 0.8 to 2.2 ml, 0.8 to 2.0 ml, 0.9 to 2.0 ml, 1.0 to 2.0 ml, etc. within the above range, but it is not limited thereto. If the volume per gram (g) is too small, a degradation rate may be too high. Further, it may be difficult to manufacture excessively large particles or the particles may not have a complete shape.
  • The porous silica particles may have a hydrophilic substituent and/or a hydrophobic substituent on an outer surface thereof and/or inside the pore. For example, only hydrophilic substituents or only hydrophobic substituents may be present on both the surface of the particle and the inside of the pore, hydrophilic substituents or hydrophobic substituents may be present on either the surface of the particle or the inside of the pore, otherwise, a hydrophilic substituent may be present on the surface of the particle while a hydrophobic substituent may be present inside of the pore, and vice versa.
  • Release of the bioactive material loaded on the porous silica particles is mainly conducted by degradation of the particles. Specifically, interaction of the porous silica particles with respect to the release environment of the bioactive material is controlled by adjusting the substituent, thereby regulating a degradation rate of the particles thus to control a release rate of the bioactive material. Further, the bioactive material may be diffused and released from the particles wherein adjusting the substituent may regulate a binding force of the bioactive material with the particles, thereby controlling the release of the bioactive material.
  • Further, for improvement of the binding force between the particles and a poorly soluble (hydrophobic) bioactive material, an additional process may be further included so that a hydrophobic substituent is present inside the pore while a hydrophilic substituent is present on the surface of the particle, in consideration of easiness in use and formulation of the composition according to the present invention.
  • The hydrophilic substituent may include, for example, aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate, polyethylene glycol, substituted or unsubstituted C1to C30 alkyl, substituted or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted C6 to C30 aryl, and C1 to C30 ester groups, etc., while the hydrophobic substituent may include, for example, substituted or unsubstituted C1to C30 alkyl, substituted or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted C6 to C30 aryl, C2 to C30 heteroaryl, halogen, C1 to C30 ester, and halogen-containing groups, etc.
  • The “substituted” functional group in the “substituted or unsubstituted” mentioned above may include at least one selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate and polyethylene glycol.
  • Further, the porous silica particles may be positively and/or negatively charged on the outer surface thereof and/ or the inside of the pore. For example, both the surface of the particle and the inside of the pore may be positively or negatively charged. Alternatively, only the surface of the particle or the inside of the pore may be positively or negatively charged. Otherwise, the surface of the particle may be positively charged while the inside of the pore may be negatively charged, and vice versa.
  • The charging may be performed, for example, by the presence of a cationic substituent or an anionic substituent.
  • The cationic substituent may include, for example, an amino group or other nitrogen-containing group as a basic group, while the anionic substituent may include, for example, a carboxyl group (—COOH), a sulfonic acid group (—SO3H), or a thiol group (—SH), etc., but it is not limited thereto.
  • Similarly, due to charging as described above, interaction between the porous silica particles with respect to the environment for releasing the bioactive material is controlled by adjusting the substituent so that a degradation rate of the particles may be regulated thus to control a release rate of the bioactive material. Further, the bioactive material may be diffused and released from the particles wherein adjusting the substituent may regulate a binding force of the bioactive material with the particles, thereby controlling the release of the bioactive material.
  • Other than the above substituents, the porous silica particles may further include another substituent, which is present on the surface of the particle and/or the inside of the pore, in order to carry a bioactive material, transfer the bioactive material to a target cell, carry a material used for other purposes or bind other additional substituents, etc., wherein the substituent may further include an antibody, a ligand, a cell permeable peptide, an aptamer, etc. coupled thereto.
  • The above-mentioned substituents, charges, coupled substances, etc. present on the surface of the particle and/or the inside of the pore may be added thereto, for example, by surface modification.
  • The surface modification may be performed, for example, by reacting a compound having a substituent to be introduced, with the particles. In this regard, the compound may include, for example, alkoxysilane having a Cl to C10 alkoxy group, but it is not limited thereto. The alkoxysilane may have at least one alkoxy group, specifically, 1 to 3 alkoxy groups, and may have a substituent to be introduced into a site in which the alkoxy group is not bonded or a substituent substituted with the alkoxy group.
  • The porous silica particles may be prepared by, for example, a small pore particle preparation and pore expansion process. If necessary, the particles may be prepared through further calcination, and surface modification processes, etc. If the particles are subjected to both the calcination and the surface modification processes, the particles may be surface-modified after the calcinations.
  • The small pore particles may be, for example, particles having an average pore diameter of 1 to 5 nm.
  • The small pore particles may be obtained by adding a surfactant and a silica precursor to a solvent and then agitating and homogenizing the solution.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether, ethyleneglycol monomethylether, ethyleneglycol monobutylether, diethyleneglycol monoethylether, diethyleneglycol monomethylether, diethyleneglycol monobutylether, propyleneglycol monomethylether, propyleneglycol monoethylether, dipropyleneglycol diethylether, triethyleneglycol monoethylether, etc.; and dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoamide, tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethan and the like. Specifically, alcohol, more specifically, methanol may be used, but it is not limited thereto.
  • When using a mixed solvent of water and an organic solvent as the solvent, a ratio of water and an organic solvent may be used in a volume ratio of, for example, 1:0.7 to 1.5, e.g., 1:0.8 to 1.3, but it is not limited thereto.
  • The surfactant may be, for example, cetyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like, and specifically, CTAB may be used.
  • The surfactant may be added in an amount of, for example, 1 to 10 g per liter of solvent, specifically, 1 to 8 g, 2 to 8 g, 3 to 8 g, etc. within the above range, but it is not limited thereto.
  • The silica precursor may be added after the agitation with addition of the surfactant to the solvent. The silica precursor may be, for example, tetramethyl orthosilicate (TMOS), but it is not limited thereto.
  • The agitation may be performed, for example, for 10 to 30 minutes, but it is not limited thereto.
  • The silica precursor may be added thereto, for example, in an amount of 0.5 to 5 ml per liter of solvent, specifically, 0.5 to 4 ml, 0.5 to 3 ml, 0.5 to 2 ml, 1 to 2 ml, etc. within the above range, but it is not limited thereto. Rather, if necessary, sodium hydroxide as a catalyst may be further used, wherein the catalyst may be added while agitating after adding the surfactant to the solvent and before adding the silica precursor to the solvent.
  • Sodium hydroxide may be used in an amount of, for example, 0.5 to 8 ml per liter of solvent, specifically, 0.5 to 5 ml, 0.5 to 4 ml, 1 to 4 ml, 1 to 3 ml, 2 to 3 ml, etc. within the above range, based on 1 M aqueous sodium hydroxide solution, but is not limited thereto.
  • After the addition of the silica precursor, the solution may be reacted with agitation. The agitation may be performed, for example, for 2 to 15 hours, specifically, 3 to 15 hours, 4 to 15 hours, 4 to 13 hours, 5 to 12 hours, 6 to 12 hours, 6 to 10 hours, etc. within the above range, but it is not limited thereto. If an agitating time (reaction time) is too short, nucleation may be insufficient.
  • After the agitation, the solution may be aged. Aging may be performed, for example, for 8 to 24 hours, specifically, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours, 10 to 14 hours, etc. within the above range, but it is not limited thereto.
  • Thereafter, the reaction product may be washed and dried to obtain porous silica particles and, if necessary, unreacted material may be isolated before washing, which may be performed, for example, by separating the supernatant through centrifugation.
  • The centrifugation may be implemented, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing may be carried out with water and/or an organic solvent. In particular, since different substances are soluble in different solvents, respectively, water and the organic solvent may be used once or several times by turns. Alternatively, water and/or the organic solvent may be used alone for washing once or several times. Such several times may include, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less, etc.
  • The organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether, ethyleneglycol monomethylether, ethyleneglycol monobutylether, diethyleneglycol monoethylether, diethyleneglycol monomethylether, diethyleneglycol monobutylether, propyleneglycol monomethylether, propyleneglycol monoethylether, dipropyleneglycol diethylether, triethyleneglycol monoethylether, etc.; and dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoamide, tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethane, etc., and, specifically, alcohol and, more specifically, ethanol may be used, but it is not limited thereto.
  • The washing may be performed under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing may be performed by filtering particles with a filter without centrifugation. The filter may include pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • For washing, water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone. The several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like.
  • The drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • Thereafter, the pores of the obtained porous silica particles may be expanded using, for example, a pore swelling agent.
  • The pore swelling agent used herein may include, for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene, etc. and, specifically, trimethylbenzene may be used, but it is not limited thereto.
  • Alternatively, the pore swelling agent used herein may be, for example, N,N-dimethylhexadecylamine (DMHA), but it is not limited thereto.
  • Pore expansion described above may be performed, for example, by mixing porous silica particles in a solvent with a pore swelling agent, and heating and reacting the mixture.
  • The solvent used herein may be, for example, water and/or an organic solvent. The organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; and, specifically, alcohol and, more specifically, ethanol may be used, but it is not limited thereto.
  • The porous silica particles may be added in a ratio of, for example, 10 to 200 g per liter of solvent, specifically, 10 to 150 g, 10 to 100 g, 30 to 100 g, 40 to 100 g, 50 to 100 g, 50 to 80 g, 60 to 80 g, etc. within the above range, but it is not limited thereto.
  • The porous silica particles may be evenly dispersed in a solvent, for example, the porous silica particles may be added to the solvent and ultrasonically dispersed therein. In the case of using a mixed solvent, the second solvent may be added after the porous silica particles are dispersed in the first solvent.
  • The pore swelling agent may be added in an amount of, for example, 10 to 200 parts by volume (vol. parts), specifically, 100 to 150 vol. parts, 10 to 100 vol. parts, 10 to 80 vol. parts, 30 to 80 vol. parts, 30 to 70 vol. parts based on 100 vol. parts of solvent within the above range, but it is not limited thereto.
  • The reaction may be performed, for example, at 120 to 180° C., specifically, 120 to 170° C., 120 to 160° C., 120 to 150° C., 130 to 180° C., 130 to 170° C., 130 to 160° C., 130 to 150° C., etc. within the above range, but it is not limited thereto.
  • The reaction may be performed, for example, for 24 to 96 hours, 2 0 specifically, 30 to 96 hours, 30 to 80 hours, 30 to 72 hours, 24 to 80 hours, 24 to 72 hours, 36 to 96 hours, 36 to 80 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 48 to 96 hours, 48 to 88 hours, 48 to 80 hours, 48 to 72 hours, etc. within the above range, but it is not limited thereto.
  • By adjusting the time and the temperature within the above ranges, respectively, the reaction may be performed sufficiently without being too much. For example, when the reaction temperature is lower, the reaction time may be increased, otherwise, when the reaction temperature is lower, the reaction time may be shortened. If the reaction is not sufficient, pore expansion may not be sufficient. On the other hand, if the reaction proceeds excessively, the particles may collapse due to the expansion of the pores.
  • The reaction may be performed, for example, while gradually increasing the temperature. Specifically, the reaction may be performed while gradually increasing the temperature at a rate of 0.5 to 15° C./min from the room temperature, specifically, 1 to 15° C./min, 3 to 15° C./min, 3 to 12° C./min, 3 to 10° C./min, etc. within the above range, but it is not limited thereto.
  • After the reaction, the reaction solution may be cooled slowly, for example, cooled by lowering the temperature step by step. Specifically, the reaction solution may be cooled by gradually decreasing the temperature at a rate of 0.5 to 20° C./min to room temperature, specifically, 1 to 20° C./min, 3 to 20° C./min, 3 to 12° C./min, 3 to 10° C./min, etc. within the above range, but it is not limited thereto.
  • After cooling, the reaction product may be washed and dried to obtain porous silica particles having expanded pores. If necessary, unreacted material may be isolated prior to washing, for example, by centrifugation to separate a supernatant.
  • The centrifugation may be performed, for example, at 6,000 to 10,000 rpm for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing may be carried out with water and/or an organic solvent. In particular, since different substances are soluble in different solvents respectively, water and the organic solvent may be used once or several times by turns. Alternatively, water and/or the organic solvent may be used alone for washing once or several times. Such several times may include, for example, two or more, ten or less, specifically, three times, 4 times, 5 times, 6 times, 7 times, 8 times, etc.
  • The organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; specifically, alcohol, more specifically, ethanol may be used, but it is not limited thereto.
  • The washing may be carried out under centrifugation, for example at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing may be performed by filtering particles with a filter without centrifugation. The filter may have pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • For washing, water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone. The several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like.
  • The drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • Thereafter, the pores of the obtained porous silica particles may be subjected to calcinations, which is a process of heating the particles to have a denser structure on the surface thereof and the inside of the pore, and removing organic materials filling the pores. For example, the calcinations may be performed at 400 to 700° C. for 3 to 8 hours, specifically, at 500 to 600° C. for 4 to 5 hours, but it is not limited thereto.
  • Then, the obtained porous silica particles may be modified on the surface and/or the inside of the pore as described above.
  • The surface modification may be performed on the surface of the particle and/or the inside of the pore. The surface of the particle and the inside of the pore may be surface-modified in the same manner or differently.
  • The particles may be charged or have hydrophilic and/or hydrophobic properties through surface modification. The surface modification may be performed, for example, by reacting a compound having a hydrophilic, hydrophobic, cationic or anionic substituent to be introduced, with the particles. In this regard, the compound may include, for example, alkoxysilane having a C1 to C10 alkoxy group, but it is not limited thereto. The alkoxysilane may have at least one alkoxy group, specifically, 1 to 3 alkoxy groups, and may have a substituent to be introduced into a site in which the alkoxy group is not bonded or a substituent substituted with the alkoxy group.
  • When the alkoxysilane is reacted with the porous silicon particles, an alkoxysilane can be bonded to the surface of the porous silica particle and/or the inside of pore through a covalent bond between a silicon atom and an oxygen atom. Further, since the alkoxysilane has a substituent to be introduced, this substituent may be introduced into the surface of the porous silica particle and/or the inside of the pore.
  • The above reaction may be performed by reacting the porous silica particles dispersed in a solvent with alkoxysilane.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, y-butyrolactone, 1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone, cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, tetramethylbenzene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.; glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether, ethyleneglycol monomethylether, ethyleneglycol monobutylether, diethyleneglycol monoethylether, diethyleneglycol monomethylether, diethyleneglycol monobutylether, propyleneglycol monomethylether, propyleneglycol monoethylether, dipropyleneglycol diethylether, triethyleneglycol monoethylether, etc.; and dimethylacetamide (DMAc), N,N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide, dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl phosphoamide, tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethan and the like. Specifically, alcohol, more specifically, methanol may be used, but it is not limited thereto.
  • The positively charging may be performed by reacting the particles with, for example, alkoxysilane having a basic group, that is, a nitrogen-containing group such as amino, aminoalkyl, etc. Specifically, N-[3-(trimethoxysilyl)propyl]ethylenediamine, N1-(3-trimethoxysilylpropy)diethylenetriamine, (3-aminopropyl)trimethoxysilane, N-[3-(trimethoxysily0propyl]aniline, trimethoxy [3-(methylamino)propyl]silane, 3-(2-aminoethylamino)propyldimethoxymethylsilane, etc. may be used, but it is not limited thereto.
  • The negatively charging may be performed by reacting the particles with, for example, alkoxysilane having an acidic group such as carboxyl, sulfonic acid, thiol, etc. Specifically, (3-mercaptopropyl)trimethoxysilane may be used, but it is not limited thereto.
  • The hydrophilic property may be obtained by reacting the particles with, for example, alkoxysilane having a hydrophilic group such as hydroxyl, carboxyl, amino, carbonyl, sulfhydryl, phosphate, thiol, ammonium, ester, imide, thioimide, keto, ether, indene, sulfonyl, polyethyleneglycol, etc. Specifically, N-[3-(trimethoxysilyl) propyl]ethylenediamine, N1-(3-trimethoxysilylpropyl)diethylenetriamine, (3-aminopropyl)trimethoxysilane, (3-mercaptopropyl)trimethoxysilane, trimethoxy [3-(methylamino)propyl]silane, 3-(2-aminoethylamino)propyldimethoxymethylsilane, etc. may be used, but it is not limited thereto.
  • The hydrophobic property may be obtained by reacting the particles with, for example, alkoxysilane having a hydrophobic substituent such as substituted or unsubstituted C1 C30 alkyl, substituted or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted C6 to C30 aryl, substituted or unsubstituted C2 to C30 heteroaryl, halogen, C1 to C30 ester, and halogen-containing groups, etc. Specifically, trimethoxy(octadecyl)silane, trimethoxy-n-octylsilane, trimethoxy(propyl)silane, isobutyl(trimethoxy)silane, trimethoxy(7-octen-1-yl)silane, trimethoxy(2-phenylethyl)silane, vinyltrimethoxysilane, cyanomethyl, 3-[(trimethoxysilyl)propyl]trithiocarbonate and (3-bromopropyl)trimethoxysilane, etc. may be used, but it is not limited thereto.
  • For improvement of the binding force between the particles and a poorly soluble (hydrophobic) bioactive material through surface modification, an additional process may be further included so that a hydrophobic substituent is present inside the pore while a hydrophilic substituent is present on the surface of the particle, in consideration of easiness in use and formulation of the composition according to the present invention. Further, a substituent for binding another material other than the bioactive material to the surface of the particle may be further provided.
  • Further, the surface modification may be performed in combination. For example, two or more surface modification may be performed on the outer surface of the particle or inside the pore. As a specific example, a compound containing a carboxyl group may be bonded to an amide-introduced silica particle through amide bond to change positively charged particles to have different surface characteristics, but it is not limited thereto.
  • The reaction of the porous silica particles with alkoxysilane may be performed, for example, under heating.
  • The heating may be performed at 80 to 180° C., for example, in a range of 80 to 160° C., 80 to 150° C., 100 to 160° C., 100 to 150° C., 110 to 150° C., etc., but it is not limited thereto.
  • The reaction of the particles with alkoxysilane may be implemented for 4 to 20 hours, for example, in a range of 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 14 hours, etc., but it is not limited thereto.
  • A reaction temperature, time, and the amount of the compound used for surface modification may be selected depending on a desired extent of surface modification. Further, varying reaction conditions depending on hydrophilicity, hydrophobicity and a charge level of the bioactive material may regulate hydrophilicity, hydrophobicity and charge level of the silica particles, thereby controlling the release rate of the bioactive material. For example, if the bioactive material has strong negative charge at neutral pH, the reaction temperature may be increased, the reaction time may be extended, or an amount of the compound to be treated may also be increased so that the porous silica particles have strong positive charge, but it is not limited thereto.
  • Further, the porous silica particles may be manufactured by, for example, preparation of small pore particles, expansion of pores, surface modification, modification of inside of the pore and the like.
  • Preparation of the small pore particles and pore expansion may be performed by the processes described above, and the washing and drying processes may be performed after the preparation of the small pore particles and after the pore expansion.
  • If necessary, isolation of the unreacted material may be preceded by washing, for example, conducted by separating the supernatant through centrifugation.
  • The centrifugation may be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing after the preparation of the small pore particles may be performed by any method under conditions within the above-illustrated range, but it is not limited thereto.
  • The washing after the pore expansion may be performed under more relaxed conditions than the above illustrative embodiments. For example, washing may be carried out three times or less, but it is not limited thereto.
  • The surface of the particle and/or the inside of the pore may be modified by the above-described method, wherein the modification may be performed in an order of the surface of the particle and then the inside of the pore, and particle washing may be further performed between the above two processes.
  • When the washing is carried out in more relaxed conditions after the preparation of small pore particles and pore expansion, the pores are filled with a reaction solution such as a surfactant used in the particle preparation and the pore expansion, such that the inside of the pore is not modified during surface modification, instead, only the surface of the particle may be modified. After then, washing the particles may remove the reaction solution in the pores.
  • Particle washing between surface modification and modification of the inside of the pore may be performed with water and/or an organic solvent. In particular, since different substances are soluble in different solvents respectively, water and the organic solvent may be used once or several times by turns. Alternatively, water and/or the organic solvent may be used alone for washing once or several times. Such several times may include, for example, two or more, ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less, etc.
  • The washing may be performed under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the above range, but it is not limited thereto.
  • The washing may be performed by filtering particles with a filter without centrifugation. The filter may include pores with a diameter of less than or equal to the diameter of the porous silica particles. If the reaction solution is filtered through such a filter, only particles remain on the filter and may be washed by pouring water and/or an organic solvent over the filter.
  • For washing, water and the organic solvent may be used once or several times by turns. Alternatively, the washing may be performed once or several times even with water or the organic solvent alone. The several times may include, for example, two or more and ten or less, specifically, three or more and ten or less, four or more and eight or less, four or more and six or less and the like. [22 6] The drying may be performed, for example, at 20 to 100° C., but it is not limited thereto. Alternatively, the drying may be performed in a vacuum state.
  • The bioactive material such as siRNA or dsRNA of the present invention may be loaded on the surface of the particle and/or the inside of the pore.
  • The loading may be implemented, for example, by mixing the porous silica particles and the bioactive material in a solvent.
  • Water and/or organic solvents may be used as the solvent, and the organic solvent used herein may include, for example: ethers such as 1,4-dioxane (particularly cyclic ethers); halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane, amyl chloride, 1,2-dibromoethane, etc.; ketones such as acetone, methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics such as benzene, toluene, xylene, etc.; alkyl amides such as N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol, propanol, butanol, etc. and the like.
  • Further, a phosphate buffered saline solution (PBS), simulated body fluid (SBF), borate-buffered saline, tris-buffered saline may be used as the solvent.
  • A ratio of the porous silica particles and the bioactive material is not particularly limited and, for example, the weight ratio may be 1:0.05 to 0.8, specifically, 1:0.05 to 0.7, 1:0.05 to 0.6, 1:0.1 to 0.8, 1:0.1 to 0.6, 1:0.2 to 0.8, 1:0.2 to 0.6, etc. within the above range.
  • The bioactive material such as siRNA or dsRNA of the present invention loaded on the porous silica particles may be gradually released over an extended period of time. Such slow release may be continuous or non-continuous, or linear or non-linear, and may vary due to the characteristics of the porous silica particles and/or interaction thereof with the bioactive material.
  • The bioactive material loaded on the porous silica particles is released while the porous silica particles are biodegraded. More particularly, the porous silica particles according to the present invention may be slowly degraded to release the loaded bioactive material in a sustained manner. Such release may be controlled by, for example, adjusting the surface area, particle diameter, pore diameter, substituents on the surface of the particle and/or the inside of pore, compactness of the porous silica particles, and the like, but it is not limited thereto.
  • In addition, the bioactive material loaded on the particles may be released while being separated from the porous silica particles and diffused, which is affected by the relationship between the porous silica particles, the bioactive material and the bioactive material releasing environment. Therefore, adjusting these conditions may control the release of bioactive material. For example, the release of bioactive material may be controlled by strengthening or weakening the binding force of the porous silica particles with the bioactive material by surface modification.
  • More particularly, if the loaded bioactive material is poorly water-soluble (hydrophobic), the surface of the particle and/or the inside of the pore may have a hydrophobic substituent to increase the binding force between the particles and the bioactive material, whereby the bioactive material may be released in a sustained manner. This may be achieved by, for example, surface modification of the particles with alkoxysilane having a hydrophobic substituent.
  • As used herein, “poorly soluble” means being insoluble (practically insoluble) or only slightly soluble (with respect to water), which is a terminology defined in “pharmaceutical Science” 18th Edition (U.S.P, Remington, Mack Publishing Company).
  • The poorly water-soluble bioactive material may have, for example, water solubility of less than 10 g/L, specifically less than 5 g/L, more specifically less than 1 g/L at 1 atmosphere and 25° C., but it is not limited thereto.
  • When the loaded bioactive material is water-soluble (hydrophilic), the surface of the particle and/or the inside of the pore may have a hydrophilic substituent to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive materials may be released in a sustained manner.
  • This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having a hydrophilic substituent.
  • The water-soluble bioactive material may have, for example, water solubility of 10 g/L or more at 1 atmosphere and 25° C., but it is not limited thereto.
  • When the loaded bioactive material is charged, the surface of the particle and/or the inside of the pore may be charged with the opposite charge thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be released in a sustained manner. This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having an acidic group or a basic group.
  • Specifically, if the bioactive material is positively charged at neutral pH, the surface of the particle and/or the inside of the pore may be negatively charged at neutral pH thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be released in a sustained manner. This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having an acidic group such as a carboxyl group (-COOH), sulfonic acid group (—SO3H), etc.
  • Further, if the bioactive material is negatively charged at neutral pH, the surface of the particle and/or the inside of the pore may be positively charged thus to increase the binding force between the porous silica particles and the bioactive material, whereby the bioactive material may be release in a sustained manner. This may be achieved by, for example, surface modification of the porous silica particles with alkoxysilane having a basic group such as an amino group, nitrogen-containing group, etc.
  • The loaded bioactive material may be released for a period of, for example, 7 days to 1 year or more depending on the type of treatment required, release environment, and porous silica particles to be used, etc.
  • Since the porous silica particles are biodegradable and may be degraded by 100%, the bioactive material loaded thereon can be released by 100%.
  • The pharmaceutical composition for preventing or treating liver cancer, which includes siRNA or dsRNA of the present invention, may further include pharmaceutically acceptable carrier and may be formulated together with the same. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit biological activities and properties of the administered compound. The pharmaceutically acceptable carrier in a composition formulated in a liquid solution is sterile and physiologically compatible, and may include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a combination of one or more of these components. Further, if necessary, other conventional additives such as antioxidants, buffers and bacteriostatic agents may also be added thereto. In addition, diluents, dispersants, surfactants, binders and lubricants may also be added so as to formulate the composition into injectable formulations such as aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets and the like.
  • The composition of the present invention is applicable in any type of formulation that contains the siRNA or dsRNA of the present invention as an active ingredient, and may be prepared in oral or parenteral formulations. Such pharmaceutical formulations of the invention may include any one suitable for oral, rectal, nasal, topical (including the cheek and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous) administration, or otherwise, may be suitable for administration through inhalation or insufflation.
  • The composition of the present invention may be administered in a pharmaceutically effective amount. An effective dose level may be determined in consideration of the type of disease, severity, activity of the drug, sensitivity to the drug, administration time, administration route and rate of release, duration of treatment, factors including concurrent drug use, and other factors well known in the medical field. The composition of the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer a minimum amount that can obtain maximum effects without side effects, which can be easily determined by those skilled in the art.
  • Dosage of the composition of the present invention may vary greatly depending on a weight, age, gender and/or health condition of a patient, diet, administration time, method of administration, excretion rate and severity of the disease. Specifically, an appropriate dosage may depend on the amount of drug accumulated in the body and/or specific efficacy of the siRNA or dsRNA of the present invention to be used. In general, the dosage may be estimated based on EC50 determined to be effective in in vivo animal models as well as in vitro. For example, the dosage may range from 0.01 μg to 1 g per kg of body weight, and the composition may be administered once or several times per unit period, in daily, weekly, monthly or yearly unit periods. Otherwise, the composition may be continuously administered for a long period of time via an infusion pump. The number of repeated doses is determined in consideration of a retention time of drug remaining in the body, a concentration of drug in the body and the like. Even after the treatment in the course of the disease treatment, the composition may be administered for preventing relapse.
  • The composition of the present invention may further include at least one active ingredient having the same or similar function in relation to treatment of liver cancer or a compound which maintains/increases solubility and/or absorbency of the active ingredient. Further, chemotherapeutic agents, anti-inflammatory agents, antiviral agents and/or immune-modulators, etc. may be optionally included.
  • In addition, the composition of the present invention may be formulated by any conventional method known in the art to provide rapid, sustained or delayed release of the active ingredient after the administration thereof to a mammal. The formulation may be in a form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • Hereinafter, the present invention will be described in detail with reference to the following examples.
    • EXAMPLE 1—Experimental Materials and Methods
  • 1. Cell Culture
  • Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7 liver cancer cell line were obtained from Korean Cell Line Bank (Seoul, Korea). All of the cell lines were cultured in EMEM (American Type Culture Collection, Manassas, Va.), RPMI-1640 or DMEM medium (Lonza, Walkersville, Md.) which is supplemented with 10% fetal bovine serum (FBS, Lonza) and 100 units/mL penicillin-streptomycin
  • (Invitrogen, Carlsbad, Calif.), in a humidified incubator at 37° C. under 5% CO2 condition.
  • 2. Synthesis and Transfection of siRNA and dsRNA
  • The siRNA and dsRNA used in this experiment were synthesized by
  • Lemonex (Seoul, Korea). Further, human BANF1, PLOD3 and SF3B4 expression plasmids subcloning gene ORF sequences (BANF1:NM_003860, PLOD3: NM_001084, SF3B4: NM_005850) in pcDNA3.1 +/C-(K)-DYK plasmid, respectively, were purchased from Genscrip™ (Piscataway, N.J., USA). Transfection was performed using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's manual.
  • 3. Extraction of RNA and DNA, and Execution of RT-PCR and qRT-PCR
  • Total RNA was isolated from frozen tissue and cells using trizol reagent (Invitrogen). 1 μg of total RNA was reverse-transcribed with cDNA by Tetro cDNA synthesis kit (Bioline, London, UK) according to the manufacturer's manual. RT-PCR reactions were conducted with nTaq DNA polymerase (Enzynomics, Taejon, Korea), followed by detection using ethidium bromide in a Gel Doc XR imaging system (Bio-Rad, Hercules, Calif.). qRT-PCR was performed by SensiFAST SYBR No-ROX Kit (Bioline) and was monitored in real time by iQ™−5 (Bio-Rad). An average Ct (threshold cycle) acquired from three replicate experiments was used for the calculation. Normalized gene expression was determined using a relative quantification method. The results were expressed as an average value of three replicate experiments. Genomic DNA from tissues and cells was isolated using DNAzol reagent (Invitrogen) according to the manual. For analysis of replication number variation, the SF3B4 genomic DNA region was amplified from 20 pairs (non-tumor and tumor) HCC tissues using a primer set from exon-1 to intron-1 according to the genome sequence of Genbank accession No. NC_000001.11. qRT-PCR was performed as described above, and glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous loading control. The primer sequences used for RT-PCR and qRT-PCR are shown in Table 5 below.
  • TABLE 5
    Accession
    Gene No. Primer Nucleotide sequence SEQ ID NO
    BANF1 NM_003860 Forward 5′- SEQ ID NO:
    GAACCGTTACGGGAACTGA 316
    A-3′
    Reverse 5′- SEQ ID NO:
    CCCGGAAGAGGTCTTCATC 317
    T-3′
    PLOD3 NM_001084 Forward 5′- SEQ ID NO:
    CAGCTCCAGGACCACTTCT 318
    C-3′
    Reverse
    5′- SEQ ID NO:
    GAGCGGGCGTAGTACTCAT 319
    C-3′
    SF3B4 NM_005850 Forward 5′- SEQ ID NO:
    CTCAGATGCAGCTTGCACA 320
    C-3′
    Reverse
    5′- SEQ ID NO:
    GGAGGGCCAGTGTATCCAT- 321
    3′
    GAPDH NM_002046 Forward 5′- SEQ ID NO:
    ACCAGGTGGTCTCCTCTGA 322
    C-3′
    Reverse
    5′- SEQ ID NO:
    TGCTGTAGCCAAATTCGTTG- 323
    3′
  • 4. Cell Growth and Proliferation Assay
  • Cell lines were seeded in 12-well plates at 30% confluence for cell growth assay. After transfection or inhibitor treatment, cells were incubated with 0.5 mg/mL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 1 h at 37° C. every 24 hours. The formazan crystal was dissolved with dimethyl sulfoxide (DMSO), and absorbance was read at 570 nm using VICTOR3™ multilabel plate reader (PerkinElmer, Boston, Mass.).
  • Cell lines were seeded in 24-well plates at 30% confluence for cell proliferation assay. After transfection, cells were treated with 5-bromo-2′-deoxyuridine (BrdU) reagent for 2 h and fixed at room temperature for 30 min. Cells were incubated with anti-BrdU antibody for 1 h at room temperature. Unbound antibody was removed by washing buffer. Horseradish peroxidase-conjugated secondary antibody was added to each well. The substrate solution was added and the reaction was stopped with stop solution after 30 min. The final product was quantified at 490 nm by VICTOR3™ multilabel plate reader (PerkinElmer).
  • 5. Cell Motility and Invasion Assay
  • For in vitro cell motility and invasion assays, Transwell plates and cell culture inserts (BD Biosciences) were used. For the coating of the invasion assay, Matrigel (BD Biosciences) was diluted to 0.3 mg/ml concentration with coating buffer (0.01 M Tris, 0.7% NaCl, pH 8.0) and 100 μl Matrigel was coated onto the upper compartment of the cell culture insert. After incubation for 1 h at 37° C., the cell culture insert was ready for seeding. After si-SF3B4 transfection, cells were appropriately seeded (0.5×105 cells/well for the motility assay, 1×105 cells/well for the invasion assay) into the cell culture insert with serum-free medium in the presence of 5% FBS as chemoattractant. After incubation for 6 h (migration assay) or 12 h (invasion assay) at 37° C., migrated or invaded cells were stained using Diff-Quik staining kit (Sysmex, Japan). Cells were photographed using an Axiovert 200 inverted microscope (Zeiss, Jena, Germany) at ×200 magnification. Cells were enumerated in three random fields of view.
  • 6. Wound Healing Assay
  • Transfected cells were seeded in wells of a 6-well plate. At 100% confluence, a scratch was made on a uniform layer using a micropipette tip. Photographs of the same area of the wound were taken after 0 and 24 h with IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).
  • 7. Mouse Liver Cancer Model
  • For xenograft tumorigenesis assay, 1×107 cells of transfected cells were mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v) Matrigel matrix (BD Biosciences). Cell suspensions were subcutaneously injected in 6-week-old male Balb/c-nude mice. Mice were examined twice per week for tumor formation at the injection sites. Tumor volumes were calculated using: 0.5×length (L)×width (W2). Each experimental group consisted of 10 mice and tumor growth was quantified by measuring tumor sizes in three orthogonal direction using calipers. Results are expressed as mean tumor volumes and 95% confidence interval. The H-ras12V activated homozygous transgenic mice were kindly provided by Dr. Dae-Yeoul Yu (Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, Daej eon, Korea). Transgenic mice were H-ras12V activated. Male mice spontaneously developed HCC beginning at 15-weeks-of-age. We surgically obtained the non-tumor region and HCC mass from five mice (35-weeks-old) and pick out three pairs of HCC tissue by pathological scoring. Diethylnitrosamine (DEN) was used to induce HCC.
  • 8. Porous Silica Particles (Mesoporous Nanoparticles) Transfection
  • siRNA specific to BANF1, PLOD3, SF3B4 was loaded in 80 μl of 3 nmol InViVojection™ RNAi-nano reagent (the porous silica nanoparticles in EXAMPLE 1-12(1)-2)-(ii), Cat. No. DHMSN-vivoRNA; Lemonex Inc., Seoul, Korea) and prepared in 200 μl of PBS. A mixture of siRNA or dsRNA and nanoparticles was intravenously injected into H-ras transgenic HCC mouse model through tail vein every week from week 9 to week 23. Sonograms (ultrasonic photographs) were taken at 17, 19, and 21 weeks by an ultrasonic machine (Affiniti 50, Philips, Seoul, Korea).
  • 9. Western Blotting Analysis
  • Cells were dissolved in a protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na2P2O7, 1 mM Na3VO4, 100×Halt protease inhibitor cocktail). Lysate containing the same amount of protein was separated by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The blots were blocked with 5% skim milk and incubated along with each antibody (Table 6).
  • TABLE 6
    Protein Manufacturer Catalog No. Dilution
    BANF1 Santa Cruz sc-33787 1:200
    PLOD3 Proteintech 11027-1-AP 1:1000
    SF3B4 Abcam ab157117 1:1000
    E-cadherin BD Biosciences 610404 1:1000
    N-cadherin BD Biosciences 610920 1:1000
    Fibronectin Santa Cruz sc-9068 1:1000
    Snail Abcam ab78105 1:1000
    Slug Cell Signaling #9585 1:500
    GAPDH Santa Cruz sc-32233 1:1000
  • 10. Statistical Analysis
  • Survival curves were plotted using the Kaplan-Meier product limit method, and significant differences between survival curves were determined using the log-rank test. All experiments were performed at least three times, and all samples were analyzed in triplicate. Results are presented as mean ±standard deviation (SD) or standard error of the mean (SEM). The statistical significance of the difference between experimental groups was assessed by paired or unpaired student's t-tests using Graphpad™ 7.0 software. Statistical significance was determined for p<0.05. Chi-square test (2-sided) was used to determine associations between parameters
  • 11. Preparation of Porous Silica Particles (Mesoporous Nanoparticles)
  • (1) Preparation of Particle 1
  • 1) Preparation of Small Pore Particles
  • A 2 L round bottom flask was charged with 960 ml of distilled water (DW) and 810 ml of MeOH. 7.88 g of CTAB was added to the flask and 4.52 ml of 1 M NaOH was rapidly added with agitating. The mixture was agitated for 10 minutes to give a homogeneously mixed solution, and 2.6 ml of TMOS was added thereto. The mixture was homogenously mixed under agitation for 6 hours and then aged for 24 hours.
  • Then, the reaction solution was centrifuged at 8000 rpm and 25° C. for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C., the product was washed five times with ethanol and distilled water by turns.
  • Thereafter, the resultant product was dried in an oven at 70° C. to obtain 1.5 g of powdery small pore silica particles (average pore diameter: 2 nm, particle diameter: 200 nm).
  • 2) Pore Expansion
  • 1.5 g of small pore silica particle powders were added to 10 ml of ethanol, followed by ultrasonic dispersion.
  • Further, 10 ml of water and 10 ml of trimethyl benzene (TMB) were added thereto, followed by ultrasonic dispersion.
  • Thereafter, the dispersion was placed in an autoclave and reacted at 160° C. for 48 hours.
  • The reaction started at 25° C. and proceeded with heating at a rate of 10° C./min, followed by cooling down at a rate of 1 to 10° C./min in an autoclave.
  • The cooled reaction solution was centrifuged at 8000 rpm and 25° C. for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • Thereafter, the resultant product was dried in an oven at 70° C. to obtain powdery porous silica particles (pore diameter: 10 to 15 nm, particle size: 200 nm).
  • 3) Calcinations
  • The porous silica particles prepared in the above 2) were placed in a glass vial and heated at 550° C. for 5 hours. After the completion of the reaction, the particles were gradually cooled to room temperature, thereby preparing particles.
  • (2) Preparation of Particle 2
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that the reaction conditions at the time of pore expansion were changed to 140° C. and 72 hours.
  • (3) Preparation of Particle 3 (10 L Scale)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 5-fold larger vessels were used and all the materials were used in a 5-fold capacity.
  • (4) Preparation of Particle 4 (Particle Diameter: 300 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 920 ml of distilled water and 850 ml of methanol were used in the preparation of small pore particles.
  • (5) Preparation of Particle 5 (Particle Diameter: 500 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 800 ml of distilled water, 1010 ml of methanol and 10.6 g of CTAB were used in the preparation of small pore particles.
  • (6) Preparation of Particle 6 (Particle Diameter: 1000 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 620 ml of distilled water, 1380 ml of methanol and 7.88 g of CTAB were used in the preparation of small pore particles.
  • (7) Preparation of Particle 7 (Pore Diameter: 4 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 2.5 ml of TMB was used at the time of pore expansion.
  • (8) Production of Particle 8 (Pore Diameter: 7 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 4.5 ml of TMB was used at the time of pore expansion.
  • (9) Preparation of Particle 9 (Pore Diameter: 17 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 11 ml of TMB was used at the time of pore expansion.
  • (10) Preparation of Particle 10 (Pore Diameter: 23 nm)
  • Porous silica particles were prepared in the same manner as in EXAMPLE 1-11(1), except that 12.5 ml of TMB was used at the time of pore expansion.
  • (11) Preparation of Particle 11 (Dual Modification)
  • 1) Preparation of Small Pore Particles
  • Small pore particles were prepared in the same manner as in EXAMPLE 1-11(1).
  • 2) Pore Expansion
  • The small pore particles were reacted with TMB in the same manner as in EXAMPLE 1-11(1)-2), cooled, and centrifuged to remove the supernatant. Thereafter, the mixture was centrifuged under the same conditions as in EXAMPLE 1-11(1)-2), washed three times with ethanol and distilled water by turns, and then dried under the same conditions as in EXAMPLE 1-11(1)-2), thereby obtaining powdery silica particles (pore diameter: 10 to 15 nm, particle diameter: 200 nm).
  • 3) Surface Modification
  • 0.8 to 1 g of porous silica particles having expanded pores were dispersed in 50 ml of toluene, and then 5 ml of (3-aminopropyl)triethoxysilane was added thereto, followed by heating at 120° C. for 12 hours under reflux. The product was subjected to the washing and drying processes described above and then dispersed along with 1 ml of triethyleneglycol (PEG3, 2-[2-(2-methoxyethoxy)ethoxy]acetic acid), 100 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 200 mg of N-Hydroxysuccinimide (NHS) in 30 ml of PBS. The dispersion was subjected to reaction with agitating at room temperature for 12 hours. Then, the resultant product was subjected to the above-described washing and drying processes.
  • Since the reaction solution in the previous step remained in the pores, the insides of the pores were not modified.
  • 4) Washing Inside of Pore
  • 800 mg of the surface-modified particle powders were dissolved in 40 ml of 2 M HCl/ethanol and refluxed with vigorous agitating for 12 hours.
  • Then, the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • Thereafter, the resultant product was dried in an oven at 70° C. to obtain powdery porous silica particles.
  • 5) Modification of Inside of Pore
  • (i) A propyl group was introduced into the pores in the same manner as in EXAMPLE 1-12(2)-1) described below.
  • (ii) An octyl group was introduced into the pores in the same manner as in EXAMPLE 1-12(2)-2) described below.
  • 12. Surface Modification of Porous Silica Particles
  • (1) Positive Charging
  • 1) Particles Having a Particle Diameter of 300 nm
  • The porous silica particles in EXAMPLE 1-11(4) were reacted with (3-aminopropyl)triethoxysilane (APTES) thus to be positively charged.
  • More particularly, 100 mg of porous silica particles were dispersed in 10 ml of toluene in a 100 ml round bottom flask provided with a bath sonicator. Then, 1 ml of APTES was added thereto, and the mixture was reacted for 12 hours with agitation at 400 rpm and 130° C.
  • After the reaction, the mixture was slowly cooled to room temperature, centrifuged at 8000 rpm for 10 minutes to remove the supernatant. During centrifugation at 8000 rpm and 25° C. for 10 minutes, the product was washed five times with ethanol and distilled water by turns.
  • Thereafter, the resultant product was dried in an oven at 70° C. to obtain powdery porous silica particles having an amino group on the surfaces of the particles and the insides of pores.
  • 2) Particles Having a Particle Diameter of 200 nm
  • (i) The porous silica particles in EXAMPLE 1-11(1) were reacted with (3-aminopropyl)triethoxysilane (APTES) thus to be positively charged, and then were modified in the same manner as in EXAMPLE 1-12(1)-1), except that 0.4 ml of APTES was added and the reaction time was changed to 3 hours.
  • (ii) The porous silica particles in EXAMPLE 1-11(9) were reacted with (3-aminopropyl)triethoxysilane(APTES) thus to be positively charged, and then were modified in the same manner as in EXAMPLE 1-12(1)-1), except that the particles prepared in EXAMPLE 1-11(9) were used.
  • (iii) The porous silica particles in EXAMPLE 1-11(10) were reacted with (3-aminopropyl)triethoxysilane(APTES) thus to be positively charged, and then were modified in the same manner as in EXAMPLE 1-12(1)-1), except that the particles prepared in EXAMPLE 1-11(10) were used.
  • (2) Introduction of Hydrophobic Group
  • 1) Propyl Group
  • The porous silica particles in EXAMPLE 1-11(1) were reacted with trimethoxy(propyl)silane to introduce a propyl group into the surfaces of the particles and the insides of the pores, and then were modified in the same manner as in EXAMPLE 1-12(1), except that 0.35 ml of trimethoxy(propyl)silane was added instead of APTES and the reaction was conducted for 12 hours.
  • 2) Octyl Group
  • The porous silica particles of EXAMPLE 1-11(1) were reacted with trimethoxy-n-octylsilane to introduce a propyl group into the surfaces of the particles and the insides of the pores, and then were modified in the same manner as in EXAMPLE 1-12(1), except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and the reaction was conducted for 12 hours.
  • (3) Negative Charging
  • 1) Carboxyl Group
  • The porous silica particles in EXAMPLE 1-11(1) were reacted with succinic anhydride thus to be negatively charged, and then were modified in the same manner as in EXAMPLE 1-12(1)-1), except that dimethyl sulfoxide (DMSO) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES, followed by agitation at room temperature for 24 hours to conduct the reaction, and DMSO was used instead of distilled water at the time of washing.
  • 2) Thiol Group
  • Modification was implemented in the same manner as in EXAMPLE 1-12(1)-1), except that 1.1 ml of MPTES was used instead of APTES.
  • 3) Sulfonic Acid Group
  • 100 mg of the porous silica nanoparticles in EXAMPLE 1-12(3)-2) were dispersed in 1 ml of 1 M aqueous sulfuric acid solution and 20 ml of 30% aqueous hydrogen peroxide, followed by agitation at room temperature to induce oxidative reaction thus to oxidize a thiol group into a sulfonic acid group. Thereafter, the product was washed and dried in the same manner as in EXAMPLE 1-12(1)-1).
  • 13. Loading of siRNA or dsRNA on Porous Silica Particles
  • 21 base pair duplex siRNA targeting green fluorescent protein (GFP) synthesized by Bionia Co. Ltd., on request was purchased from the same
  • (Sequence: sense;
    5′-GGCUACGUCCAGGAGCGCACC-3′, (SEQ ID NO: 324)
    antisense;
    5′-UGCGCUCCUGGACGUAGCCUU-3′ (SEQ ID NO: 325)).
  • After mixing 10 μg of the porous silica particles in EXAMPLE 1-12(1)-2)-(ii) and 50 pmol of siRNA were mixed in 1×PBS condition and allowed to be loaded at room temperature for 30 minutes.
    • EXAMPLE 2—Analysis of Inhibitory Rate of Indicator Gene Expression by siRNA or dsRNA of the Present Invention
  • According to the experimental procedures in EXAMPLE 1-1 to 3, indicator genes of siRNA and dsRNA of the present invention (BANF1 variant 1, BANF1 variant 2, PLOD3, and SF3B4) were analyzed, and the results are shown in Tables 7 to 10 below.
  • Referring to Tables 7 to 10 below, it can be seen that all the siRNAs and dsRNAs of the present invention could inhibited the expression of the indicator genes at high inhibitory rates.
  • TABLE 7
    Verification of validity of siRNA, dsRNA for inhibition
    of human BANF1, transcript variant 1, mRNA (Gene Bank
    number: NM_003860.3) expression
    Base SEQ ID NO Expression inhibitory rate (%)
    5 87.73
    6 79.64
    7 82.3
    8 76.21
    9 89.6
    10 83.42
    11 73.18
    12 85.44
    13 69.57
    14 77.3
    15 82.92
    16 91.38
    17 84.11
    18 88.36
    19 87.83
    20 67.72
    21 82.29
    22 63.23
    23 76.24
    24 87.7
    25 62.57
    26 72.92
    27 65.58
    28 72.91
  • TABLE 8
    Verification of validity of siRNA, dsRNA for inhibition
    of human BANF1, transcript variant 2, mRNA (Gene Bank
    number: NM_001143985.1) expression
    Base SEQ ID NO Expression inhibitory rate (%)
    29 92.55
    30 91.49
    31 86.44
    32 77.1
    33 73.82
    34 76.6
    35 88.33
    36 82.53
    37 81.64
    38 68.4
    39 79.72
    40 91.6
    41 87.37
    42 53.77
    43 86.39
    44 68.63
    45 83.22
    46 78.16
    47 73.48
    48 68.3
    49 85.27
    50 88.74
    51 92.32
    52 74.8
    53 84.31
    54 64.9
    55 74.72
  • TABLE 9
    Verification of validity of siRNA, dsRNA for
    inhibition of human PLOD3 gene sequence (Gene Bank
    number: NM_001084.4) expression
    Base SEQ ID NO Expression inhibitory rate (%)
    56 87.62
    57 78.13
    58 92.72
    59 83.49
    60 86.8
    61 64.29
    62 73.33
    63 85.83
    64 82.68
    65 76.92
    66 91.64
    67 85.77
    68 79.1
    69 82.4
    70 84.63
    71 89.26
    72 76.4
    73 76.8
    74 68.27
    75 77.44
    76 86.26
    77 84.3
    78 81.52
    79 79.35
    80 76.63
    81 85.32
    82 62.72
    83 64.3
    84 77.13
    85 83.78
    86 86.71
    87 82.33
    88 68.46
    89 74.88
    90 72.7
    91 83.69
    92 85.3
    93 76.62
    94 82.11
    95 83.46
    96 71.25
    97 72.73
    98 87.6
    99 69.91
    100 81.38
    101 78.27
    102 74.8
    103 69.76
    104 62.45
    105 87.22
    106 82.7
    107 74.5
    108 86.25
    109 83.7
    110 74.13
    111 76.29
    112 73.52
    113 82.86
    114 73.52
    115 73.71
    116 82.55
    117 69.72
    118 77.8
    119 86.49
    120 71.3
  • TABLE 10
    Verification of validity of siRNA, dsRNA for
    inhibition of Human SF3B4 gene sequence (Gene Bank
    number: NM_005850.4) expression
    Base SEQ ID NO Expression inhibitory rate (%)
    121 83.71
    122 81.83
    123 87.62
    124 86.39
    125 78.64
    126 82.7
    127 84.25
    128 74.11
    129 63.36
    130 76.25
    131 74.32
    132 92.19
    133 84.72
    134 81.3
    135 83.4
    136 88.63
    137 78.25
    138 85.1
    139 81.68
    140 83.57
    141 78.33
    142 72.45
    143 76.72
    144 81.36
    145 83.2
    146 72.41
    147 73.64
    148 86.77
    149 84.8
    150 82.56
    151 73.12
    152 78.66
    153 82.5
    154 76.63
    155 62.95
    156 89.6
    157 77.2
    • EXAMPLE 3—Identification of Excellent RNA Delivery by Porous Silica Particles
  • With respect to Hepa-lcic7 and SNU-449 cell lines in EXAMPLE 1, siRNAs, each of which includes a sense RNA having a sequence shown in Table 11 below and an antisense RNA having a complementary sequence thereto, were subjected to in vitro transfection by the methods described in EXAMPLE 1-2 or 1-8, respectively. Then, expression levels of the corresponding markers of the above siRNAs were measured by Western blotting, and the results are shown in FIG. 1.
  • Referring to FIG. 1, when the siRNAs shown in Table 11 were transfected, it can be seen that the markers were effectively inhibited. Specifically, when siRNA was loaded on the porous silica particles and then transfected, the expression inhibitory rate was demonstrated to be higher.
  • TABLE 111
    Sense RNA Name in
    SEQ ID NO sequence FIG. 1 Target gene
    SEQ ID NO: 311 5′- Banf1 Mouse BANF1 gene
    CCUCAGCGUUUCAAU
    CUUUUU-3′
    SEQ ID NO: 312 5′- Plod3 Mouse PLOD3 gene
    CGACUGCAGAAUCUC
    CUCUUU-3′
    SEQ ID NO: 313 5′- Sf3b4 Mouse SF3B4 gene
    CUGCUUUACGAUACU
    UUCAUU-3′
    SEQ ID NO: 314 5′- Control —
    CCUACGCCACCAAUU
    UCGU-3′
    SEQ ID NO: 28 5′- BANF1 Human BANF1 gene
    AAGAAGCUGGAGGAA
    AGGGGUUU-3′
    SEQ ID NO: 119 5′- PLOD3 Human PLOD3 gene
    GCAUCUGGAGCUUUC
    UGUA UU-3′
    SEQ ID NO: 136 5′- SF3B4 Human SF3B gene
    GCAGUACCUCUGUAA
    CCGU UU-3′
    • EXAMPLE 4—Identification of Liver Cancer Cell Metastatic Potential Inhibition by siRNA or dsRNA of the Present Invention
  • 1 Cell Motility and Invasion Assay and Wound Healing Assay
  • With respect to SNU-449 cell line in EXAMPLE 1-1, siRNAs, each of which includes a sense RNA having a sequence shown in Table 12 below and an antisense RNA having a complementary sequence thereto, were subjected to in vitro transfection by the method described in EXAMPLE 1-2. Migration and invasion of markers corresponding to the above siRNAs were analyzed by the method in EXAMPLE 1-5, while a scratch wound healing ability was analyzed by the method in EXAMPLE 1-6, and the analyzed results are shown in FIG. 2.
  • TABLE 12
    SEQ ID NO Sense RNA sequence Name in FIG. Target gene
    SEQ ID NO: 314 5′- Control —
    CCUACGCCACCAAUUUC
    GU-3′
    SEQ ID NO: 28 5′- BANF1 Human BANF1
    AAGAAGCUGGAGGAAAG gene
    GGGUUU-3′
    SEQ ID NO: 119 5′- PLOD3 Human PLOD3
    GCAUCUGGAGCUUUCUG gene
    UA UU-3′
    SEQ ID NO: 136 5′- SF3B4 Human SF3B4
    GCAGUACCUCUGUAACC gene
    GU UU-3′
  • Referring to A of FIG. 2, it can be seen that, as compared to the control group, the cells with the markers, which were knockdown by transfection of siRNAs listed in Table 12, exhibited significantly reduced migration and invasion. Referring to B of FIG. 2, it was observed that, as compared to the control group, wound-healing ability was considerably decreased. These results demonstrate that the siRNA or dsRNA of the present invention can inhibit metastatic potential of liver cancer cells, while reducing malignant progression.
  • 2. Identification of EMT Regulatory Proteins Inhibition
  • In order to identify whether siRNA or dsRNA of the present invention can inhibit indicator actors for expression of N-cadherin, Fibronectin, Snail and Slug, which are representative epithelial-mesenchymal transition (EMT) regulatory proteins in relation to the metastasis of liver cancer cells, which in turn can inhibit metastasis of liver cancer, siRNAs, each of which includes a sense RNA having the sequence shown in Table 12 above and an antisense RNA having the complementary sequence thereto, were subjected to in vitro transfection to the SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2. Then, expression levels of the markers corresponding to the siRNAs and expression levels of the EMT regulatory proteins above were analyzed by the method in EXAMPLE 1-9, and the analyzed results are shown in A of FIG. 3.
  • Referring to A of FIG. 3, it can be seen that, as compared to the control group, the cells with the markers, which were knockdown by transfection of siRNAs listed in Table 12, exhibited that the expression levels of N-cadherin, Fibronectin, Snail and Slug, which are epithelial-mesenchymal transition (EMT) regulatory proteins, as well as the expression levels of the markers are inhibited. These results demonstrate that the siRNA or dsRNA of the present invention may selectively inhibit the expression of the corresponding markers, thereby inhibiting metastatic potential of liver cancer cells.
    • EXAMPLE 5—Identification of Tumor Growth Inhibition by siRNA or dsRNA of the Present Invention
  • siRNAs, each of which includes a sense RNA having the sequence shown in Table 12 above and an antisense RNA having the complementary sequence thereto, were subjected to in vitro transfection to the SNU-449 cell line in EXAMPLE 1 by the method described in EXAMPLE 1-2. Thereafter, athymic nude mice were subjected to subcutaneous injection of the transfected cells, followed by analyzing sizes of hepatic tumors and survival rates of the mice, and the analyzed results are shown in B of FIG. 3.
  • Referring to the left image of B of FIG. 3, it can be seen that most of the experimental groups have a significantly smaller hepatic tumor size than the control group. Therefore, it is understood that knockdown of the markers by the siRNA or dsRNA of the present invention may reduce overall tumor growth rate and decrease the average tumor volume.
  • Referring to the right image of B of FIG. 3, it can be seen that the tumor-free survival rates of the experimental groups are significantly higher than that of the control group. Specifically, when 50 days elapses after the subcutaneous injection of the transfected cells, the control group exhibited tumors in 6 mice of 10 mice whereas the experimental group exhibited tumors in 1 to 2 mice only among 10 mice, thereby indicating that the siRNA or dsRNA of the present invention may effectively inhibit the growth of hepatic tumors.
    • EXAMPLE 6—Identification of liver cancer prevention efficacy of siRNA or dsRNA of the present invention
  • siRNAs, each of which includes a sense RNA having a sequence shown in Table 13 below and an antisense RNA having a complementary sequence thereto, were subjected to in vivo transfection by the method described in EXAMPLE 1-8, and processes thereof, ultrasonic images and the number of tumors over time are shown in A of FIG. 4. Further, expression inhibitory levels for the indicator genes by the siRNAs loaded on the porous nanoparticles were analyzed by the method in EXAMPLE 1-9 and shown in B of FIG. 4.
  • Referring to A of FIG. 4, in the case of the control group injected with only porous nanoparticles, multiple large hepatic tumors were found in 3 to 4 mice 17 weeks after the injection whereas the experimental group injected with porous nanoparticles loaded with siRNA exhibited that only a relatively small hepatic tumor was found in 2 to 4 mice 19 weeks after the injection. Further, as shown in B of FIG.
  • 4, it can be seen that, as compared to the control group, the experimental group could significantly reduce the expression levels of the indicator gens in vivo as a result of Western blotting. These results demonstrate that the siRNA or dsRNA of the present invention may effectively inhibit the expression of the markers in vivo, and exert excellent effects in inhibition of hepatic tumor formation and prevention of liver cancer.
  • TABLE 13
    SEQ Sense RNA Name in
    ID NO sequence FIG. 1 Target gene
    SEQ ID
    5′- Banfl Mouse BANF1
    NO: 311 CCUCAGCGUUUCAAUCU gene
    UUUU-3′
    SEQ ID
    5′- Plod3 Mouse PLOD3
    NO: 312 CGACUGCAGAAUCUCCU gene
    CUUU-3′
    SEQ ID
    5′- 5f3b4 Mouse 5F3B4
    NO: 313 CUGCUUUACGAUACUUU gene
    CAUU-3′
    • EXAMPLE 7—Identification of Formation of Porous Silica Particles and Pore Expansion
  • The small pore particles and the prepared porous silica particles in EXAMPLE 1-11(1) to (3) were observed with a microscope to identify whether the small pore particles were uniformly formed and the pores were sufficiently expanded thus to uniformly form the porous silica particle (FIGS. 5 to 8).
  • FIG. 5 is photographs illustrating the porous silica particles in EXAMPLE 1-11(1) and FIG. 6 is photographs illustrating the porous silica particles in EXAMPLE 1-11(2), demonstrating that the porous silica particles in a spherical shape with sufficiently expanded pores were uniformly formed. Further, FIG. 7 is photographs illustrating the small pore particles in EXAMPLE 1-11(1), and FIG. 8 is comparative photographs illustrating the small pore particles in EXAMPLEs 1-11(1) and 1-11(3), demonstrating that spherical small pore particles were uniformly formed.
    • EXAMPLE 8—Identification of Biodegradability of Porous Silica articles
  • In order to identify the biodegradability of the porous silica particles in EXAMPLE 1-11(1), the degree of biodegradation at 37° C. under SBF (pH 7.4) was observed with a microscope at 0 hours, 120 hours and 360 hours, and the observed results are shown in FIG. 9.
  • Referring to FIG. 9, it can be seen that the porous silica particles were almost completely degraded after 360 hours elapse from the observation.
    • EXAMPLE 9—Measurement of Absorbance Ratio of Porous Silica Particles
  • 1. Measurement Method
  • The absorbance ratio according to the following Equation 1 was measured:

  • At/A0   [Equation 1]
  • (wherein A0 is absorbance of the porous silica particles measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particles into a cylindrical dialysis membrane having pores with a diameter of 50 kDa,
  • 15 ml of the same solvent as the suspension is placed outside the dialysis membrane while being in contact with the dialysis membrane, followed by horizontal agitation at 60 rpm and 37° C. inside and outside the dialysis membrane, and
  • At is absorbance of the porous silica particles measured after t hours elapses from the measurement of A0).
  • Specifically, 5 mg of the porous silica particle powders were dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in a dialysis membrane having a pore diameter of 50 kDa shown in FIG. 10. 15 ml of SBF was added to an outer membrane, and SBF of the outer membrane was replaced every 12 hours. Degradation of the porous silica particles was performed while horizontally agitating at 37° C. and 60 rpm.
  • Thereafter, the absorbance was measured by UV-vis spectroscopy and analyzed at λ=640 nm. [431]
  • 2. Absorbance Ratio Measurement Results
  • The absorbance ratio of the porous silica particles in EXAMPLE 1-11(1) was 2 5 measured according to the above method, and the results are shown in FIG. 11.
  • Referring to FIG. 11, it can be seen that t when the absorbance ratio becomes ½ was about 58 hours, demonstrating very slow degradation.
  • 3. Measurement Results by Particle Size
  • The absorbance of the porous silica particles in each of EXAMPLEs 1-11(1), (5) and (6) was measured according to Equation 1 above and the results are shown in FIG. 12 (using SBF as a suspension and a solvent).
  • Referring to FIG. 12, it can be seen that t is decreased as the particle diameter is increased.
  • 4. Measurement Results to Pore Diameter Average Diameter
  • The absorbance of the porous silica particles in each of EXAMPLEs 1-11(1) and (9), and the small pore silica particles in EXAMPLE 1-11(1) as a control group was measured according to the above Equation 1 (using SBF as suspension and solvent).
  • Referring to FIG. 13, it can be seen that the porous silica particles in the examples have significantly larger t than the control.
  • 5. Measurement Results by pH
  • The absorbance of the porous silica particles in EXAMPLE 1-11(4) was measured by pH. The absorbance was measured in SBF and in Tris at pH 2, 5 and 7.4, respectively, and the results are shown in FIG. 14.
  • Referring to FIG. 14, there was a difference in t by pH, but t when all absorbance ratios became ½ was 24 or more.
  • 6. Charging
  • The absorbance of the porous silica particles in EXAMPLE 1-12(1)-1) was measured, and the results are shown in FIG. 15 (using Tris (pH 7.4) as a suspension and a solvent).
  • Referring to FIG. 15, even the positively charged particles showed that t when the absorbance ratio of the absorbance became ½ was 24 or more.
    • EXAMPLE 10—Release of siRNA or dsRNA Loaded on Porous Silica Particles
  • 10 μl of the porous silica particles loaded with the siRNA in EXAMPLE 1-13 was resuspended in SBF (pH 7.4, 37° C.) and placed in a dialysis membrane having a pore diameter of 20 kDa (the tube in FIG. 16). Then, the dialysis tube was immersed in 1.5 ml of SBF. The release of siRNA was performed while horizontally agitating at 37° C. and 60 rpm.
  • The release solvent was recovered at 0.5, 1, 2, 4, 8, 12, 24 hours prior to 24 hours and then, every 24 hours, 0.5 ml of the release solvent was recovered for fluorescence measurement and SBF was added thereto.
  • The fluorescence intensity of the siRNA was measured at a wavelength of 670 nm (λex=647 nm) to determine the degree of siRNA release, and the results are shown in FIG. 17.
  • Referring to FIG. 17, it can be seen that the time at which 50% of the siRNA was released was about 40 hours or more.
    • EXAMPLE 11—Identification of Target Delivery of siRNA or dsRNA Loaded on Porous Silica Particles
  • In order to verify whether the siRNAs of the present invention can play a role of a transporter in a desired level for study of siRNA delivery in animal level, tumor inhibitory rates due to the release of bioactive material in mice (rats) were confirmed.
  • Specifically, Balb/c nude male mice (5 weeks old) were purchased from Orient Bio Inc., and 3 million HeLa cells (cervical cancer cells) were dispersed in sterilized 1×PBS to proliferate Xenograft tumors subcutaneously injected into the mice. When 70 mm3 size of solidified tumors were observed, PBS, FITC-porous silica particles (porous silica particles in EXAMPLE 1-12(1)-2)-(ii)), and FITC-porous silica particles loaded with siRNA (porous silica particles in EXAMPLE 1-12(1)-2)-(ii)) were injected into tumors in the mice, respectively. Then, fluorescence intensities and distribution thereof were measured immediately before, immediately after, and 48 hours after the administration, by means of FOBI fluorescence in vivo imaging system (Neo science, Korea).
  • FITC labeling were implemented by: dispersing 50 mg of silica particles in 1 ml of dimethyl sulfoxide (DMSO); adding 25 μg (10 μl) of FITC-NHS (N-hydroxysuccinimide) solution (2.5 mg/mL) thereto; reacting the mixture at room temperature for 18 hours while shielding light with aluminum foil; purifying the reaction product through centrifugation (8500 rpm, 10 minutes); discarding the supernatant while collecting settled particles; and evenly dispersing the particles in ethanol, wherein the above processes were repeated three and four times with ethanol and distilled water to purify until FITC color is invisible in the supernatant.
  • Referring to FIG. 18 demonstrating results of the above experiments, the control group refers to administration of PBS alone, Cy5-siRNA refers to administration of siRNA in EXAMPLE 1-13 alone, FITC-DDV refers to administration of FITC-labeled porous silica particles alone, and the complex refers to administration of FITC-labeled porous silica particles loaded with the siRNA in EXAMPLE 1-13. Referring to this figure, it can be seen that the siRNA loaded on the particles and delivered into the body has a longer duration of activity and stays longer at the injected site, thereby exhibiting strong fluorescence even after 48 hours.
  • A sequence listing electronically submitted with the present application on Jan. 28, 2020 as an ASCII text file named 20200128_Q25720LC02_TU_SEQ, created on Jan. 28, 2020 and having a size of 124,000 bytes, is incorporated herein by reference in its entirety.

Claims (15)

1. A pharmaceutical composition for preventing or treating liver cancer, comprising at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 157, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 310.
2. The composition according to claim 1, wherein the composition comprises at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 12, 14 to 19, 21, 23, 24, 26, 28 to 34, 35 to 37, 39 to 41, 43, 45 to 47, 49 to 53, 55 to 60, 62 to 73, 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 to 157, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 165, 167 to 172, 174, 176, 177, 179, 181 to 187, 188 to 190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to 226, 228 to 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310.
3. The composition according to claim 1, wherein the composition comprises at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 5 to 28, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 158 to 181.
4. The composition according to claim 1, wherein the composition comprises at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 29 to 55, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 182 to 208.
5. The composition according to claim 1, wherein the composition comprises at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 56 to 120, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 209 to 273.
6. The composition according to claim 1, wherein the composition comprises at least one of the followings:
siRNA which includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 121 to 157, and an antisense RNA having a complementary sequence thereto; and
dsRNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 274 to 310.
7. The composition according to claim 1, wherein the siRNA or dsRNA is loaded on at least one carrier selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin, lipid particles, polyethylene glycol, poly-L-guluronic alginate, polyglycolic-polyacticpolyactic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and hydrogel.
8. The composition according to claim 7, wherein the carrier is a porous silica particle characterized in that t when a ratio of absorbance in the following Equation 1 becomes ½ is 20 or more:

At/A0   [Equation 1]
wherein A0 is absorbance of the porous silica particle measured by placing 5 ml of a suspension including 1 mg/ml of the porous silica particle into a cylindrical dialysis membrane having pores with a diameter of 50 kDa;
15 ml of the same solvent as the suspension is placed outside the dialysis membrane while being in contact with the dialysis membrane, followed by horizontal agitation at 60 rpm and 37° C. inside and outside the dialysis membrane; and
At is absorbance of the porous silica particle measured after t hours elapses from the measurement of Ao.
9. The composition according to claim 8, wherein the t is 40 or more.
10. The composition according to claim 8, wherein the siRNA includes a sense RNA having at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 28, 119 and 136, and an antisense RNA having a complementary sequence thereto; and
the dsRNA has at least one sequence selected from the group consisting of sequences of SEQ ID NOs: 181, 272 and 289.
11. The composition according to claim 8, wherein the porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
12. The composition according to claim 8, wherein the porous silica particle has at least one hydrophilic substituent selected from the group consisting of aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether, indene, sulfonyl, methylphosphonate, polyethylene glycol, substituted or unsubstituted C1 to C30 alkyl, substituted or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted C6 to C30 aryl, and C1 to C30 ester groups.
13. The composition of claim 8, wherein the porous silica particle is positively or negatively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
14. The composition of claim 8, wherein the porous silica particle is positively charged on an outer surface of the particle or an inside of a pore thereof at neutral pH.
15. The composition of claim 8, wherein the porous silica particle has an average particle diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
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