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WO2008069621A1 - Nouvelle utilisation de gènes mig12 et pio5 - Google Patents

Nouvelle utilisation de gènes mig12 et pio5 Download PDF

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Publication number
WO2008069621A1
WO2008069621A1 PCT/KR2007/006366 KR2007006366W WO2008069621A1 WO 2008069621 A1 WO2008069621 A1 WO 2008069621A1 KR 2007006366 W KR2007006366 W KR 2007006366W WO 2008069621 A1 WO2008069621 A1 WO 2008069621A1
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WIPO (PCT)
Prior art keywords
gene
nucleotide sequence
seq
sirna
cancer
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/KR2007/006366
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English (en)
Inventor
Mi Sun Won
Kyung-Sook Chung
Young-Joo Kim
Shin-Jung Choi
Dong Myung Kim
Nam Hui Yim
Ji-Won Ahn
Cheol-Goo Hur
Kyung Bin Song
Hee Gu Lee
Moon Hee Kim
Young Il Yeom
Eun Young Song
Hyung Ju Lee
Kyeong-Eun Jung
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Korea Research Institute of Bioscience and Biotechnology KRIBB
Samchully Pharmaceutical Co Ltd
Original Assignee
Korea Research Institute of Bioscience and Biotechnology KRIBB
Samchully Pharmaceutical Co Ltd
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Priority claimed from KR1020070126815A external-priority patent/KR100986465B1/ko
Publication of WO2008069621A1 publication Critical patent/WO2008069621A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC

Definitions

  • the present invention relates to a composition for cancer diagnosis or anticancer drug screening, comprising an MIG 12 gene or MIG 12 gene-expressed protein, a composition for cancer diagnosis or anticancer drug screening, comprising an OIP5 gene or OIP5 gene- expressed protein, a composition for treatment of cancer, comprising an inhibitor of the aforesaid genes or an inhibitor of the aforesaid proteins and a pharmaceutically acceptable carrier, and a cancer diagnosis kit comprising at least one of an MIG 12 gene, an OIP5 gene, and an MIG12- or OIP5-expressed protein.
  • pombe a powerful model organism to investigate cancer-related genes.
  • the overexpression of a heterologous gene may cause growth defects or morphological changes by interfering in the cell cycle or basic metabolic pathways, which can be used as a simple indication of a gene functioning in the dysregulation of basic cellular processes.
  • the overexpression of human heterologous genes in S. pombe, which produces observable phenotypes, can provide insights into cellular functions relevant to cancer. Therefore, the investigation of the overexpression effects of the heterologous human gene is a powerful method to find clues associated with carcinogenesis.
  • RNAi is a naturally occurring process in eukaryotes by which double-stranded RNAs
  • RNA interference is an efficient method of identifying targets for gene therapy in a variety of diseases.
  • RNAi has become a widespread tool for reverse genetics in invertebrate model systems and has rapidly emerged as a novel therapeutic approach to treat human diseases.
  • a short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference for a relatively long time.
  • shRNA which consists of siRNA sense sequence, and 3-10 bases of linker-antisense siRNA sequence, uses a vector introduced into cells and utilizes the U 6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA.
  • shRNAs can also be cloned into lentivirus or adenovirus and used for in vivo animal study.
  • OIP5 (GenBank NM_007280) was identified as Opa (Neisseria gonorrhoeae opacity- associated)-interacting protein. OIP5 is homologous to OIP4, which is a protein antigen that is expressed in tumors and also in some normal tissues, including testes and endometrium. OIP5, a cancer-testis specific gene, was confirmed to be up-regulated in the tumor tissue compared to normal tissue in gastric cancer. It was suggested that OIP5 may be a novel immunotherapy target for patients with gastric cancer. Recently, OIP5 was reported as LINT-25 since it interacted with LAP2 ⁇ , lamina associated polypeptide 2 ⁇ .
  • LINT-25 protein levels were up- regulated during Gl phase in proliferating cells and upon cell cycle exit in quiescence, senescence and differentiation.
  • LINT-25 accumulated inheterochromatin foci and transient expression of LINT-25 in proliferating cells caused loss of LAP2 ⁇ and subsequent cell death.
  • MIG 12 (GenBank NM_021242) was isolated as the gene whose protein product interacts with Midi. Migl2 is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development. Endogenous Migl2 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization. When co- transfected with Midi, Migl2 is massively recruited to thick filamentous structures composed of tubulin. These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays. The relevance of MIG 12 to cancer has not been reported.
  • the present invention has been made in view of the above problems, and it is an object of the present invention to provide a novel use of MIG 12 and OIP5 genes.
  • an object of the present invention is to provide a composition for diagnosing cancer or for screening anticancer drugs comprising an MIG 12 gene or a protein expressed from the MIG12 gene; a composition for diagnosing cancer or for screening anticancer drugs comprising an OIP5 gene or a protein expressed from the OIP5 gene; a composition for preventing or treating cancer comprising an inhibitor of the aforesaid genes or an inhibitor of the aforesaid proteins and a pharmaceutically acceptable carrier; and a kit for diagnosing cancer comprising one or more selected from an MIG 12 gene, an OIP5 gene, a protein expressed from MIG 12 gene, and a protein expressed from OIP5 gene.
  • composition for diagnosing cancer comprising an MIG 12 or OIP5 gene.
  • MIG 12 gene or “OIP5 gene” refers to a full-length DNA or RNA of the MIG 12 or OIP5 genes, or a portion or variant thereof.
  • composition of the present invention may further comprise buffer or distilled water to stably maintain a structure of nucleic acid.
  • the expression level of MIGl 2 gene or OIP5 gene is specifically increased in cancer cells or cancer cell lines.
  • the diagnosis of cancer can be performed by analysis of the overexpression of MIG 12 gene or OIP5 gene, using the genes of the present invention.
  • an MIG12 or OIP5 gene for diagnosing cancer and a method for diagnosing cancer, comprising confirming the reaction between a sample obtained from a subject and an MIG 12 or OIP5 gene.
  • DNA-DNA DNA-RNA or DNA- protein
  • DNA chip DNA chip
  • protein chip PCR (polymerase chain reaction)
  • Northern blotting Southern blotting
  • ELISA Enzyme Linked Immunosorbent assay
  • yeast two-hybrid assay 2-D gel electrophoresis, in vitro binding assay, etc.
  • incidence of cancer in individuals can be determined by examining whether a gene of interest is expressed at a high level in a subject, through hybridization of an intact or partial gene as a probe with the nucleic acid isolated from a body fluid of the subject, and detection of a hybridization product according to any conventional method known in the art, such as reverse transcription-polymerase chain reaction (RT-PCR), Southern blotting, Northern blotting, and the like.
  • RT-PCR reverse transcription-polymerase chain reaction
  • Southern blotting Southern blotting
  • Northern blotting and the like.
  • compositions for diagnosing cancer comprising a protein expressed from an MIG 12 gene or OIP5 gene.
  • the composition of the present invention may further comprise buffer or distilled water to stably maintain a structure of the protein.
  • the expression level of MIG 12 or OIP5 gene is specifically increased in cancer cells. Therefore, the presence of cancer can be diagnosed by analysis of the overexpression of MIG 12 or OIP5 gene or the corresponding protein, using the protein expressed from the MIG 12 gene or OIP5 gene.
  • confirmation of the reaction between the subject-derived sample and the protein may be carried out by using any conventional methods used in the cases of DNA-protein, RNA-protein or protein-protein, for example, DNA chip, protein chip, PCR (polymerase chain reaction), Northern blotting, Southern blotting, Western blotting, ELISA (Enzyme Linked Immunosorbent assay), histoimmunostaining, yeast two-hybrid assay, 2-D gel electrophoresis, in vitro binding assay, etc.
  • the analysis of individual carcinogenesis can be carried out by examining whether a gene of interest is expressed at a high level in a subject, through hybridization of the gene-expressed intact or partial protein as a probe with the nucleic acid or protein isolated from a body fluid of the subject, and detection of a hybridization product according to any conventional method known in the art, such as reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and the like.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the probe is labeled with a suitable label material such as radioisotopes, enzymes, the like, it is possible to readily confirm the presence of the target gene.
  • composition of the present invention may comprise an antibody specific for the aforesaid protein, instead of that protein.
  • an antibody specific for the aforesaid protein instead of that protein.
  • amounts of the proteins expressed from these genes also increase. Therefore, as disclosed in Example 1 which will follow, when antibodies directed against the proteins expressed from these genes are used, it is possible to diagnose cancer by detection of the protein through an antigen-antibody reaction.
  • Monoclonal antibodies directed against the aforesaid protein may be produced by any conventional method for production of monoclonal antibodies known in the art or otherwise may be commercially available. Quantitative analysis of monoclonal antibodies against the aforesaid proteins may be generally carried out by color development using secondary antibodies conjugated with a certain enzyme (such as alkaline phosphatase (AP) or horseradish peroxidase (HRP)) and substrates thereof, or otherwise may be carried out using the protein- specific monoclonal antibodies which are directly conjugated with AP or HRP enzymes. Alternatively, instead of monoclonal antibodies, it may also be possible to use polyclonal antibodies which recognize the aforesaid protein. These polyclonal antibodies may be obtained by any conventional method for production of antiserum known in the art.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • the present invention provides a composition for screening anticancer drugs, comprising an MIG12 or OIP5 gene. Further, the present invention provides a use of the same composition for screening anticancer drugs, and a method for screening anticancer drugs, comprising contacting a test material with the composition as a target material, and confirming the reaction between the target material and the test material to determine whether the test material activates or suppresses the expression of the MIG12 or OIP5 gene.
  • the reaction between the composition and the test material can be confirmed by any conventional method used in the cases of DNA-DNA, DNA-RNA, DNA-protein, DNA-compound.
  • a hybridization test to confirm binding of the gene to the test material in vitro
  • a method of determining a gene expression level involving reacting of mammalian cells with the test material, and subjecting the reaction product to Northern blot analysis, quantitative PCR, or quantitative real-time PCR and a method of determining an expression level of a reporter gene involving linking the reporter gene to the gene of interest, introducing the resulting gene construct into a cell and reacting the cell with a test material.
  • the composition of the present invention may further comprise distilled water or buffer to stably maintain a structure of the nucleic acid, in addition to the aforesaid gene.
  • the present invention provides a method for screening anticancer drugs, comprising inducing phenotypic changes of a model cell or animal cell using the aforesaid composition as a target material and determining whether the test material activates or suppresses the expression of the gene.
  • candidate materials having direct inhibitory effects on the gene expression and protein function may be screened by cell-based analysis through phenotype assay (including growth rate, morphological changes and temperature sensitivity) using a model organism or animal cell with overexpression of the aforesaid gene.
  • the present invention provides a composition for screening anticancer drugs, comprising a protein expressed from an MIG 12 or OIP5 gene. Further, the present invention provides a use of the same composition for screening anticancer drugs and a method for screening anticancer drugs, comprising a test material with the aforesaid composition as a target material, and confirming the reaction between the target material and the test material to determine whether the test material activates or suppresses the function of the protein.
  • the reaction between the composition and the test material can be confirmed by any conventional method used in the cases of protein-protein, DNA-compound and the like.
  • a method of determining activity of a test material after reaction of the protein with the test material yeast two-hybrid assay, detection of phage-displayed peptide clones bound to the aforesaid protein, high throughput screening (HTS) using natural products and chemical libraries, drug hit HTS (HTS), cell-based screening, and DNA array screening.
  • the composition of the present invention may further comprise buffer or reaction liquid to stably maintain a structure or physiological activity of the corresponding protein.
  • the composition of the present invention may comprise cells expressing the aforesaid protein, or cells harboring a plasmid that expresses the aforesaid protein under a promoter capable of controlling a transcription rate.
  • the test material may be a material which is suspected to have the possibility of being a cancer metastasis inhibitor according to a conventional criteria and strategy for selection, or may be a material which is randomly selected, such as individual nucleic acids, proteins, extracts or natural products, compounds, and the like.
  • test materials There may be two types of test materials which are obtained through the screening method of the present invention: a material capable of enhancing the gene expression or protein function and a material capable of inhibiting the gene expression or protein function.
  • the former material may be an anticancer drug candidate via development of an inhibitor against the test material, whereas the latter material may be directly an anticancer drug candidate.
  • These anticancer drug candidates will serve as a leading compound during a subsequent development process of anticancer drugs.
  • a novel anticancer drug can be created by structural modification and optimization of the leading compound such that the leading compound can exhibit inhibitory effects on function of the gene or expression products thereof.
  • siRNA of an MIGl 2 gene hereinafter, referred to as MIG 12 siRNA or siMIG12
  • siRNA of an OIP5 gene hereinafter, referred to as OIP5 siRNA or siOIP5
  • siRNA is prepared by simultaneous synthesis of a sense strand of the gene and an anti- sense strand exhibiting practical gene expression inhibition in cells. Thereafter, two strands synthesized are allowed to form a double-stranded RNA, followed by intracellular incorporation.
  • siRNA sequence of the present invention refers to a sense strand of a gene of interest.
  • a candidate siRNA sequence region of the MIG 12 gene may have a nucleotide sequence consisting of 19-30 contiguous nucleotides as set forth in SEQ ID NO: 14, 15, 16, 17, or 18.
  • the nucleotide sequence of SEQ ID NO: 14, 15, 16, 17, or 18 is a partial sequence of the MIG 12 gene (NM_021242) of SEQ ID NO: 1 and has a nucleotide sequence consisting of from nucleotide 845 to nucleotide 950, a nucleotide sequence consisting of from nucleotide 970 to nucleotide 1030, a nucleotide sequence consisting of from nucleotide 1080 to nucleotide 1160, a nucleotide sequence consisting of from nucleotide 1220 to nucleotide 1290, or a nucleotide sequence consisting of from nucleotide 1320 to nucleotide 1400, each sequence comprising 61-106 nucleotides
  • the MIG 12 siRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 14, 15, 16, 17 and 18.
  • the MIG 12 siRNA may have a nucleotide sequence of SEQ ID NO: 3 or 4.
  • the sequence of SEQ ID NO: 3 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1331 to nucleotide 1349 of the MIG12 gene of SEQ ID NO: 1 and contains two overhang nucleotides of TT
  • the sequence of SEQ ID NO: 4 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1123 to nucleotide 1141 of the MIGl 2 gene of SEQ ID NO: 1 and contains two overhang nucleotides of TT.
  • These sequences may have superior gene expression inhibition (RNAi effects) on the anti-sense strand complementary to the sequence of SEQ ID NO: 3 or 4.
  • a candidate siRNA sequence region of the OIP5 gene may have a nucleotide sequence consisting of 19-30 contiguous nucleotides as set forth in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26.
  • the nucleotide sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26 is a partial sequence of the OIP5 gene (NM_007280) of SEQ ID NO: 2 and has a nucleotide sequence consisting of from nucleotide 1 to nucleotide 60, a nucleotide sequence consisting of from nucleotide 120 to nucleotide 180, a nucleotide sequence consisting of from nucleotide 380 to nucleotide 460, a nucleotide sequence consisting of from nucleotide 518 to nucleotide 580, a nucleotide sequence consisting of from nucleotide 720 to nucleotide 780, a nucleotide sequence consist consisting of from
  • the OIP5 siRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25, and 26.
  • the OIP5 siRNA may have a nucleotide sequence of SEQ ID NO: 5 or 6.
  • the sequence of SEQ ID NO: 5 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 426 to nucleotide 444 of the OIP5 gene of SEQ ID NO: 2 and contains two overhang nucleotides of TT
  • the sequence of SEQ ID NO: 6 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 417 to nucleotide 435 of the OIP5 gene of SEQ ID NO: 2 and contains two overhang nucleotides of TT.
  • siRNA of the present invention may be chemically modified in order to prevent rapid degradation which may occur by the action of in vivo nucleases.
  • siRNA has a double- stranded structure, and therefore is relatively stable as compared to single-stranded ribonucleic acid or antisense oligonucleotide molecules.
  • siRNA is readily susceptible to in vivo nuclease degradation, so it is possible to lower a degradation rate of siRNA via chemical modification thereof.
  • Chemical modification of siRNA to secure that siRNA is chemically stable and resistant against rapid degradation may be carried out by any conventional method well known to those skilled in the art.
  • boranophosphate or phosphorothioate modification The most conventional method used in chemical modification of siRNA is boranophosphate or phosphorothioate modification. These materials form stable nucleoside-nucleoside association of siRNA molecules, thereby conferring resistance to nucleic acid degradation. Even though it is resistant to nucleic acid degradation, the boranophosphate-modified ribonucleic acid is not synthesized by a chemical reaction and is synthesized only by incorporation of boranophosphate into ribonucleic acid via in vitro transcription.
  • the boranophosphate modification is a relatively easy method, but has a disadvantage associated with difficulty of site-directed modification at a certain position of target molecules or compounds.
  • the phosphorothioate modification is advantageous for introduction of sulfur elements into a desired site, but excessive phosphothioation may result in problems associated with decreased efficiency, toxicity, and formation of non-specific RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • chemical modification of a ribose ring is also known to enhance the nuclease resistance of nucleic acid. Particularly, modification at 2'-postion of the ribose ring leads to stabilization of siRNA molecules.
  • siRNA molecules increases only with correct introduction of a methyl group into the above-specified position of the ribose ring. Further, introduction of excessively large numbers of methyl groups may result in loss of ribonucleic acid-mediated interference. Chemical modifications may also be made to improve the in vivo pharmacokinetic retention time and efficiency (Mark et al., Molecular Therapy, 13:644-670, 2006).
  • siRNA of the present invention may be incorporated in the form of a complex with a nucleic acid delivery system into a pharmaceutical composition for treating cancer.
  • the nucleic acid delivery system for intracellular delivery of nucleic acid materials may be broadly divided into a viral vector system and a non-viral vector system.
  • the most conventionally and widely used system is a viral vector system because it provides a high delivery efficiency and a long retention time in vivo.
  • a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and the like are widely employed.
  • viral vector systems are efficient in intracellular delivery of ribonucleic acid, but suffer from various problems associated with safety concerns, such as difficulty of recombination of the ribonucleic acid into a virus having in vivo activity, elicitation of immune response, undesirable random insertion of the ribonucleic acid into host chromosomes, and the like.
  • the non-viral vector system exhibits various advantages, such as low toxicity and immune response, feasibility of repeated administration, convenient formation of a complex of the delivery system with ribonucleic acid, and easy industrial-scale production, as compared to the viral vector system.
  • organ/cell-targeted nucleic acid delivery to affected cells or tissue lesions can be carried out by conjugation of a specific ligand with the non- viral vector.
  • the non- viral vector that can be used in the present invention may include various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles, and the like.
  • the nucleic acid delivery system can significantly enhance delivery efficiency of the desired nucleic acid into animal cells and can deliver nucleic acid into any type of animal cells, depending upon the desired uses and applications of the nucleic acids.
  • siRNA of the present invention is a short double-stranded RNA (dsRNA) consisting of 19-30 bp nucleotides.
  • siRNA of the present invention can be used for functional study of related genes.
  • the present invention provides an shRNA of an MIG 12 (hereinafter, referred to as MIG12 shRNA or shMIG12) or an shRNA of OIP5 gene (hereinafter, referred to as OIP5 shRNAor shOIP5).
  • shRNA small hairpin RNA
  • shRNA which consists of siRNA sense sequence and 3-10 bases of linker-antisense siRNA sequence, uses a plasmid vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed.
  • the loop sequence of the resulting shRNA is cleaved by Dicer, an
  • shRNAs can also be cloned into lenti virus or adenovirus and used for in vivo animal study. shRNA exhibits
  • the MIG 12 shRNA may also comprise a nucleotide sequence corresponding to an niRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 14, 15, 16, 17 and 18 of the MIG12 gene.
  • the MIG 12 shRNA may comprise an RNA nucleotide sequence of SEQ ID NO: 4 and an antisense RNA sequence which is linked via a 3-10 bp loop to and is reverse complementary to the sequence of SEQ ID NO: 4.
  • the RNA nucleotide sequence of SEQ ID NO: 4 is a 19-bp nucleotide sequence which corresponds to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1123 to nucleotide 1141 of the MIGl 2 gene of SEQ ID NO: 1 and may have superior gene expression inhibition (RNAi effects) on an anti- sense strand reverse complementary to the sequence of SEQ ID NO: 4.
  • RNA nucleotide sequence refers to an RNA nucleotide sequence part with exception of a short sequence consisting of two bases (TT) from the nucleotide sequence of SEQ ID NO: 4.
  • a top strand for construction of an expression vector to express the MIG 12 shRNA may have a sequence as set forth in SEQ ID NO: 27.
  • the OIP5 shRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25, and 26.
  • the OIP5 shRNA may comprise an RNA nucleotide sequence of SEQ ID NO: 5 and an antisense RNA sequence which is linked via a 3-10 bp loop to and is reverse complementary to the sequence of SEQ ID NO: 5.
  • the RNA nucleotide sequence of SEQ ID NO: 5 is a 19-bp nucleotide sequence which corresponds to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 426 to nucleotide 444 of the OIP5 gene of SEQ ID NO: 2 and may have superior gene expression inhibition (RNAi effects) on an anti-sense strand reverse complementary to the sequence of SEQ ID NO: 5.
  • RNA nucleotide sequence refers to an RNA nucleotide sequence part with exception of a short sequence consisting of two bases (TT) from the nucleotide sequence of SEQ ID NO: 5.
  • a top strand for construction of an expression vector to express the OIP5 shRNA may have a sequence as set forth in SEQ ID NO: 28.
  • shRNA small hairpin RNA
  • siRNA small hairpin RNA
  • US Patent Application Nos. 2004/106567 Al and 2004/0086884 disclose a variety of delivery mechanisms, such as viral and non- viral vectors comprising shRNA, liposome delivery systems, in vivo injection systems of plasmid DNAs, artificial viral envelope and polylysine conjugates, as well as plenty of information about the expression constructs/vectors.
  • the present invention provides a composition for treating cancer, comprising an inhibitor of an MIG 12 or OIP5 gene.
  • inhibitor encompasses a gene-expression inhibitor.
  • composition may further comprise one or more pharmaceutically acceptable carriers.
  • the gene of the present invention is expressed at a high level in cancer cells, and administration of an inhibitor against the aforementioned gene (hereinafter, referred to as "gene inhibitor”) can suppress the gene expression to thereby inhibit incidence of cancer.
  • the present invention provides a use of a gene inhibitor for treating cancer, and a method for treating cancer, comprising administering a therapeutically effective amount of the gene inhibitor to a subject in need thereof.
  • term “treating cancer” or “treatment of cancer” or “cancer treatment” encompasses prevention and inhibition of cancer.
  • the gene inhibitor may be an antisense oligonucleotide for mRNA of the MIGl 2 or OIP5 gene.
  • the antisense oligonucleotide has been successfully employed to achieve gene-specific inhibition both in vivo and in vitro.
  • the antisense oligonucleotide is a short synthetic DNA strand (or DNA analog) which is antisense (or complementary) to a certain DNA or RNA target.
  • the antisense oligonucleotide is proposed to prevent expression of the protein encoded by a DNA or RNA target.
  • the antisense oligonucleotide binds to the target to thereby halt the protein expression at a transcription, translation or splicing stage.
  • antisense oligonucleotide has been successfully used in cell culture and animal models of disease (Hogrefe, 1999). Additional modification of antisense oligonucleotides to secure that oligonucleotide is more stable and resistant against in vivo nuclease degradation may be carried out by any conventional method well known to those skilled in the art.
  • the term "antisense oligonucleotide” encompasses double-stranded DNAs (dsDNAs) or single- stranded DNAs (ssDNAs), dsRNAs or ssRNAs, DNA/RNA hybrids, DNA and RNA analogs, and oligonucleotides having modifications of base, sugar, and/or backbone.
  • the oligonucleotide may be chemically modified to improve the stability thereof and increase the resistance against nuclease degradation, according to a conventional method known in the art. These modification methods are conventionally known in the art, and may include, but are not limited to, modifications of the oligonucleotide backbone, sugar moieties and bases.
  • the gene inhibitor may be siRNA (Small Interfering RNA) of the MIG 12 or OIP5 gene.
  • siRNA is prepared by simultaneous synthesis of a sense strand of the gene and an anti- sense strand exhibiting practical gene expression inhibition in cells. Thereafter, two strands synthesized are allowed to form a double-stranded RNA, followed by intracellular incorporation.
  • siRNA sequence of the present invention refers to a sense strand of a gene of interest.
  • a candidate siRNA sequence region of the MIG 12 gene may have a nucleotide sequence consisting of 19-30 contiguous nucleotides as set forth in SEQ ID NO: 14, 15, 16, 17, or 18.
  • the nucleotide sequence of SEQ ID NO: 14, 15, 16, 17, or 18 is a partial sequence of the MIG12 gene (NM_021242) of SEQ ID NO: 1 and has a nucleotide sequence consisting of from nucleotide 845 to nucleotide 950, a nucleotide sequence consisting of from nucleotide 970 to nucleotide 1030, a nucleotide sequence consisting of from nucleotide 1080 to nucleotide 1160, a nucleotide sequence consisting of from nucleotide 1220 to nucleotide 1290, or a nucleotide sequence consisting of from nucleotide 1320 to nucleotide 1400, each sequence comprising 61-106 nucleotides
  • a nucleotide sequence complementary to the aforesaid nucleotide sequence may be a sequence which is capable of efficiently inducing RNAi.
  • the MIGl 2 siRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 14, 15, 16, 17 and 18.
  • the MIG 12 siRNA may have a nucleotide sequence of SEQ ID NO: 3 or 4.
  • the sequence of SEQ ID NO: 3 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1331 to nucleotide 1349 of the MIG12 gene of SEQ ID NO: 1 and contains two overhang nucleotides of TT
  • the sequence of SEQ ID NO: 4 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1123 to nucleotide 1141 of the MIG 12 gene of SEQ ID NO: 1 and contains two overhang nucleotides of TT.
  • These sequences may have superior gene expression inhibition (RNAi effects) on the anti-sense strand complementary to the sequence of SEQ ID NO: 3 or 4.
  • a candidate siRNA sequence region of the OIP5 gene may have a nucleotide sequence consisting of 19-30 contiguous nucleotides as set forth in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26.
  • the nucleotide sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 26 is a partial sequence of the OIP5 gene (NM_007280) of SEQ ID NO: 2 and has a nucleotide sequence consisting of from nucleotide 1 to nucleotide 60, a nucleotide sequence consisting of from nucleotide 120 to nucleotide 180, a nucleotide sequence consisting of from nucleotide 380 to nucleotide 460, a nucleotide sequence consisting of from nucleotide 518 to nucleotide 580, a nucleotide sequence consisting of from nucleotide 720 to nucleotide 780, a nucleotide sequence consist consisting of from
  • the OIP5 siRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25, and 26.
  • the 0IP5 siRNA may have a nucleotide sequence of SEQ ID NO: 5 or 6.
  • the sequence of SEQ ID NO: 5 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 426 to nucleotide 444 of the OIP5 gene of SEQ ID NO: 2 and contains two overhang nucleotides of TT
  • the sequence of SEQ ID NO: 6 may be a sequence which has a 19-bp nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 417 to nucleotide 435 of the OIP5 gene of SEQ ID NO: 2 and contains two overhang nucleotides of TT.
  • siRNA of the present invention may be chemically modified in order to prevent rapid degradation which may occur by the action of in vivo nucleases.
  • siRNA has a double- stranded structure, and therefore is relatively stable as compared to single-stranded ribonucleic acid or antisense oligonucleotide molecules.
  • siRNA is readily susceptible to in vivo nuclease degradation, so it is possible to lower a degradation rate of siRNA via chemical modification thereof.
  • Chemical modification of siRNA to secure that siRNA is chemically stable and resistant against rapid degradation may be carried out by any conventional method well known to those skilled in the art.
  • boranophosphate or phosphorothioate modification The most conventional method used in chemical modification of siRNA is boranophosphate or phosphorothioate modification. These materials form stable nucleoside-nucleoside association of siRNA molecules, thereby conferring resistance to nucleic acid degradation. Even though it is resistant to nucleic acid degradation, the boranophosphate-modified ribonucleic acid is not synthesized by a chemical reaction and is synthesized only by incorporation of boranophosphate into ribonucleic acid via in vitro transcription.
  • the boranophosphate modification is a relatively easy method, but has a disadvantage associated with difficulty of site-directed modification at a certain position of target molecules or compounds.
  • the phosphorothioate modification is advantageous for introduction of sulfur elements into a desired site, but excessive phosphothioation may result in problems associated with decreased efficiency, toxicity, and formation of non-specific RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • chemical modification of a ribose ring is also known to enhance the nuclease resistance of nucleic acid. Particularly, modification at 2'-postion of the ribose ring leads to stabilization of siRNA molecules.
  • siRNA molecules increases only with correct introduction of a methyl group into the above-specified position of the ribose ring. Further, introduction of excessively large numbers of methyl groups may result in loss of ribonucleic acid-mediated interference. Chemical modifications may also be made to improve the in vivo pharmacokinetic retention time and efficiency (Mark et al, Molecular Therapy, 13:644-670, 2006).
  • siRNA of the present invention may be incorporated in the form of a complex with a nucleic acid delivery system into a pharmaceutical composition for treating cancer.
  • the nucleic acid delivery system for intracellular delivery of nucleic acid materials may be broadly divided into a viral vector system and a non-viral vector system.
  • the most conventionally and widely used system is a viral vector system because it provides a high delivery efficiency and a long retention time in vivo.
  • a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and the like are widely employed.
  • viral vector systems are efficient in intracellular delivery of ribonucleic acid, but suffer from various problems associated with safety concerns, such as difficulty of recombination of the ribonucleic acid into a virus having in vivo activity, elicitation of immune response, undesirable random insertion of the ribonucleic acid into host chromosomes, and the like.
  • the non-viral vector system exhibits various advantages, such as low toxicity and immune response, feasibility of repeated administration, convenient formation of a complex of the delivery system with ribonucleic acid, and easy industrial-scale production, as compared to the viral vector system.
  • organ/cell-targeted nucleic acid delivery to affected cells or tissue lesions can be carried out by conjugation of a specific ligand with the non- viral vector.
  • the non- viral vector that can be used in the present invention may include various formulations such as liposomes, cationic polymers, micelles, emulsions, nanoparticles, and the like.
  • the nucleic acid delivery system can significantly enhance delivery efficiency of the desired nucleic acid into animal cells and can deliver nucleic acid into any type of animal cells, depending upon the desired uses and applications of the nucleic acids.
  • siRNA of the present invention is a short double-stranded RNA (dsRNA) consisting of
  • siRNA of the present invention can be used for functional study of related genes.
  • the gene inhibitor may be shJRNA of an MIGl 2 or OIP5 gene.
  • shRNA small hairpin RNA
  • shRNA which consists of siRNA sense sequence and 3-10 bases of linker-antisense siRNA sequence, uses a plasmid vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed.
  • the loop sequence of the resulting shRNA is cleaved by Dicer, an RNase III enzyme, to generate mature siRNA which will exhibit RNAi effects.
  • shRNAs can also be cloned into lentivirus or adenovirus and used for in vivo animal study.
  • the MIG 12 shRNA exhibits RNAi effects for a relatively long time, as compared to siRNA. Similar to MIG 12 siRNA, the MIG 12 shRNA may also comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 14, 15, 16, 17 and 18 of the MIGl 2 gene.
  • the MIG12 shRNA may comprise an RNA nucleotide sequence of SEQ ID NO: 4 and an antisense RNA sequence which is linked via a 3-10 bp loop to and is reverse complementary to the sequence of SEQ ID NO: 4.
  • the RNA nucleotide sequence of SEQ ID NO: 4 may comprise an RNA nucleotide sequence of SEQ ID NO: 4 and an antisense RNA sequence which is linked via a 3-10 bp loop to and is reverse complementary to the sequence of SEQ ID NO: 4.
  • NO: 4 is a 19-bp nucleotide sequence which corresponds to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 1123 to nucleotide 1141 of the MIG 12 gene of
  • SEQ ID NO: 1 may have superior gene expression inhibition (RNAi effects) on an anti- sense strand reverse complementary to the sequence of SEQ ID NO: 4.
  • RNA nucleotide sequence refers to an RNA nucleotide sequence part with exception of a short sequence consisting of two bases (TT) from the nucleotide sequence of SEQ ID NO: 4.
  • a top strand for construction of an expression vector to express the MIG 12 shRNA may have a sequence as set forth in SEQ ID NO: 27.
  • the OIP5 shRNA may comprise a nucleotide sequence corresponding to an mRNA sequence derived from a nucleotide sequence of 19 to 30 contiguous nucleotides, among one or more sequences selected from SEQ ID NOS: 19, 20, 21,
  • the OIP5 shRNA may comprise an RNA nucleotide sequence of SEQ ID NO: 5 and an antisense RNA sequence which is linked via a 3-10 bp loop to and is reverse complementary to the sequence of SEQ ID NO: 5.
  • the RNA nucleotide sequence of SEQ ID NO: 5 is a 19-bp nucleotide sequence which corresponds to an mRNA sequence derived from a nucleotide sequence ranging from nucleotide 426 to nucleotide 444 of the OIP5 gene of SEQ ID NO: 2 and may have superior gene expression inhibition (RNAi effects) on an anti-sense strand reverse complementary to the sequence of SEQ ID NO: 5.
  • RNA nucleotide sequence refers to an RNA nucleotide sequence part with exception of a short sequence consisting of two bases (TT) from the nucleotide sequence of SEQ ID NO: 5.
  • a top strand for construction of an expression vector to express the OIP5 shRNA may have a sequence as set forth in SEQ ID NO: 28.
  • Expression constructs/vectors comprising shRNA (small hairpin RNA) having the siRNA nucleotide sequence to enhance the intracellular retention and transfection effects of siRNA are known to those skilled in the art. For example, US Patent Application Nos.
  • 2004/106567 Al and 2004/0086884 disclose a variety of delivery mechanisms, such as viral and non- viral vectors comprising shRNA, liposome delivery systems, in vivo injection systems of plasmid DNAs, artificial viral envelope and polylysine conjugates, as well as plenty of information about the expression constructs/vectors.
  • siRNA has a promising potential as a very strong inhibitor drug on in vivo expression of a certain gene, in view of the long retention time upon cell culture and under in vivo conditions, in vivo transfection capacity and resistance against serum degradation (Bertrand et al., 2002).
  • siRNA even at a relatively low concentration can achieve inhibitory effects on gene expression comparable to or higher than those obtained by the antisense oligonucleotide and therefore is proposed as a substitute for the antisense oligonucleotide (Thompson, 2002).
  • siRNAs are widely utilized for inhibition of gene expression in animal models of disease.
  • the antisense oligonucleotide, siRNA, or shRNA of the present invention which is used for treatment of cancer, may be administered in the form of a composition further comprising pharmaceutically acceptable carrier(s).
  • Suitable pharmaceutically acceptable carriers may include water, saline, PBS (phosphate buffered saline), dextrin, glycerol, and ethanol. These materials may be used alone or in any combination thereof. Additionally, the composition of the present invention may be appropriately formulated by a conventional method known in the art, such that it is possible to achieve fast, sustained or delayed release of active ingredients after administration of the composition to a subject including mammals or humans.
  • the gene inhibitor may be antisense oligonucleotides, siRNAs, or shRNAs, as well as any material capable of inhibiting expression of a gene of interest, such as low-molecular weight compounds, natural products, bioactive proteins, in vivo proteins, novel proteins, and synthetic and naturally-occurring chemicals. Therefore, it is possible to use compounds known as the inhibitor of the aforementioned gene in the art, and materials identified by a screening method using the same gene. Further, the present invention provides a composition for treating cancer, comprising an inhibitor against the protein expressed from an MIG12 or OIP5 gene (hereinafter, referred to as "protein inhibitor").
  • protein inhibitor an inhibitor against the protein expressed from an MIG12 or OIP5 gene
  • Suppression of the gene of the present invention results in subsequent inhibition of the corresponding protein expression to thereby inhibit development and/or progress of cancer. Consequently, it is possible to inhibit development of cancer via suppression of expression of the corresponding protein from the MIG 12 or OIP5 gene.
  • the present invention provides a use of a protein inhibitor for treating cancer, and a method for treating cancer, comprising administering a therapeutically effective amount of the same protein inhibitor to a subject in need thereof.
  • term “treating cancer” or “treatment of cancer” or “cancer treatment” encompasses prevention and inhibition of cancer.
  • the protein inhibitor may be an antibody directed against the protein expressed from the gene of the present invention.
  • Monoclonal antibodies directed against the aforesaid protein may be produced by any conventional method for production of monoclonal antibodies known in the art or otherwise may be commercially available.
  • polyclonal antibodies instead of monoclonal antibodies, it may also be possible to use polyclonal antibodies which recognize the aforesaid protein. These polyclonal antibodies may be obtained by any conventional method for production of antiserum known in the art.
  • the composition of the present invention may be formulated into a variety of dosage forms by further inclusion of one or more pharmaceutically acceptable carriers in combination with the above-mentioned active ingredient.
  • the pharmaceutically acceptable carrier may be comprised of a minimum amount of an additive to improve a shelf life or effectiveness of a binding protein, such as a wetting agent, emulsif ⁇ er, preservative or buffer.
  • the anticancer composition of the present invention may be used in combination with one or more anticancer drugs.
  • the anticancer composition may further comprise chemotherapeutic agents well-known to those skilled in the art, for example, alkylating agents such as cyclophosphamide, aziridine, alkyl alkone sulfonate, nitrosourea, dacarbazine, carboplatin, cisplatin, and the like, antibiotics such as mitomycin C, anthracycline, doxorubicin (Adriamycin), and the like, antimetabolic agents such as methotrexate, 5- fluorouracil (5-FU) and cytarabine, plant-derived agents such as Vinca alkaloids, and hormones.
  • chemotherapeutic agents well-known to those skilled in the art, for example, alkylating agents such as cyclophosphamide, aziridine, alkyl alkone sulfonate, nitrosourea
  • composition of the present invention may further comprise pharmaceutically and physiologically acceptable additives, in addition to the active ingredient.
  • additives may include excipients, disintegrating agents, sweeteners, binders, coating agents, blowing agents, lubricants, glidants, solubilizers, etc.
  • composition of the present invention may further comprise one or more pharmaceutically acceptable carriers besides the active ingredient, to be formulated to a pharmaceutical composition appropriately.
  • a pharmaceutically acceptable carrier which is sterile and biocompatible may be used such as saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. These materials may be used alone or in any combination thereof. If necessary, other conventional additives may be added such as antioxidants, buffers, bacteriostatic agents, and the like.
  • compositions may be additionally added to the composition to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions, or oral formulations such as pills, capsules, granules, and tablets.
  • injectable formulations such as aqueous solutions, suspensions, and emulsions
  • oral formulations such as pills, capsules, granules, and tablets.
  • the composition may be preferably formulated into a desired dosage form, depending upon diseases to be treated and ingredients, using any appropriate method known in the art, as disclosed in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA.
  • Dosage forms of the composition of the present invention may include granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juice, suspensions, emulsions, drops or injectable liquid formulations and sustained-release formulations of active ingredient(s).
  • the pharmaceutical composition can be administered via a conventional route, for example intravenously, intraarterially, intraperitoneally, intramuscularly, intrathoracically, percutaneously, intranasally, locally, rectally, orally, intraocularly, intradermally, or by inhalation.
  • a conventional route for example intravenously, intraarterially, intraperitoneally, intramuscularly, intrathoracically, percutaneously, intranasally, locally, rectally, orally, intraocularly, intradermally, or by inhalation.
  • the term "therapeutically effective amount” refers to an amount of an active ingredient that is required to exert anticancer effects.
  • the effective dose of the active ingredient in accordance with the present invention may vary depending upon various factors such as kinds of disease, severity of disease, kinds and contents of active ingredients and other components contained in the composition, kinds of dosage forms, age, weight, health, sex and dietary habits of patients, administration times and routes, release rates of the composition, treatment duration, and co-administered drugs.
  • the dosage is preferably in a range of 0.01 ng/kg to 10 mg/kg for siRNA, 0.01 ng/kg to 10 mg/kg for shRNA, 0.01 ng/kg to 10 mg/kg for antisense oligonucleotide to mRNA of the aforesaid gene, 0.1 ng/kg to 10 mg/kg for a compound, 0.1 ng/kg to 10 mg/kg for a monoclonal antibody directed against the aforesaid protein, 0.1 ng/kg to 10 mg/kg for a low-molecular weight compound, 0.1 ng/kg to 10 mg/kg for a natural product, and 0.1 ng/kg to 10 mg/kg for bioactive proteins, respectively.
  • the present invention provides a kit for cancer diagnosis, comprising at least one of an MIG12 gene, an OIP5 gene, and a protein expressed from the MIG12 or OIP5 gene.
  • the cancer diagnosis kit may further comprise a material for application of DNA chips, protein chips, etc., which are used to examine the reaction of the composition with a test sample.
  • the MIG 12 gene may have a nucleotide sequence of SEQ ID NO: 1 or its variant sequence with deletion, substitution or insertion of one or more bases.
  • the OIP5 gene may have a nucleotide sequence of SEQ ID NO: 2 or its variant sequence with deletion, substitution or insertion of one or more bases.
  • the term "substantial sequence identity” or “substantial sequence homology” is used to indicate that a sequence of interest exhibits substantial structural or functional equivalence with another sequence. Differences may be due to inherent variations in codon usage among different species, for example. Structural differences are considered de minimis if there is a significant amount of sequence overlap or similarity between two or more different sequences or if the different sequences exhibit similar physical characteristics even if the sequences differ in length or structure.
  • MIG12 and OIP5 genes of the present invention Upon overexpression of MIG12 and OIP5 genes of the present invention in model cells, these genes are implicated in cell cycle regulation and growth factors, are expressed at high levels in cancer cell lines, and increase a cell growth rate. Therefore, inhibition of expression of these genes leads to suppression of cancer cell growth. For these reasons, MIG12 and OIP5 genes can be used as a target gene for diagnosis or cancer therapy of various cancers.
  • the present invention will be used for research and development of personalized anticancer drugs having low adverse side effects, such as cancer target gene-specific therapeutic agents, for example biopharmaceuticals such as siRNA/shRNA drugs, antibody drugs and protein drugs, and cancer-targeted drugs such as low-molecular weight synthetic drugs and natural products which target MIG 12 and OIP5 proteins. Further, the present invention will contribute to the development of a source technology which forms the basis of study of new cancer-related mechanisms.
  • FIG. 1 illustrates a schematic diagram of cloning a human gene from a plasmid (Entry clone) with insertion of the human gene into a destination vector, S. pombe;
  • FIG. 2 illustrates phenotypic changes observable upon overexpression of a human gene MIG12 in S. pombe.
  • FIG. 2A Cell growth inhibition
  • FIG. 2B Morphological changes of cells
  • FIG. 2C Intracellular changes of DNA levels
  • FIG. 2D Confirmation of human gene expression
  • FIG. 3 illustrates phenotypic changes observable upon overexpression of a human gene OIP5 in S. pombe.
  • FIG. 3A Cell growth inhibition
  • FIG. 3B Morphological changes of cells
  • FIG. 3C Intracellular changes of DNA levels
  • FIG. 3D Confirmation of human gene expression
  • FIG. 4 illustrates RT-PCR measurement of mRNA levels for MIGl 2 and OIP5 genes in liver and stomach cancer cell lines;
  • FIG. 5 illustrates comparison of expression levels of an OIP5 gene in cancer tissues and normal tissues of colorectal cancer patients
  • FIG. 6 illustrates comparison of expression levels of an OIP5 mRNA in cancer tissues of colorectal cancer patients, using RT-PCR
  • FIG. 7 illustrates a graph for the results of proliferation assay after expression of MIG 12 and OIP5 genes in animal cells (NIH3T3), as experiments for prediction of the relationship between genes and carcinogenesis;
  • FIG. 8 illustrates energy profiles of MIG12 and OIP5 genes
  • FIG. 9 illustrates a graph showing a degree of cell growth inhibition in various stomach, liver, lung and colorectal cancer cell lines, as the results of siRNA experiments to confirm the potentiality of MIG 12 and OIP5 genes as a cancer target gene
  • FIG. 10 illustrates cell photographs showing cell growth inhibition in a variety of stomach and liver cancer cell lines, as the results of siRNA experiments to confirm the potentiality of MIG12 and OIP5 genes as a cancer target gene;
  • FIG. 11 illustrates FACS analysis results for measurement of cell apoptosis due to expression inhibition of the corresponding gene after siRNA experiments
  • FIG. 12 illustrates changes in expression levels of apoptosis-related genes through RNAi experiments using siMIG12 or siOIP5;
  • FIG. 13 illustrates shRNA design (A) and experimental results (B);
  • FIG. 14 illustrates a graph showing cell growth inhibition and inhibition degree after overexpression of MIG12 and OIP5 genes in fission yeast S. pombe as a model cell
  • FIG. 15 illustrates investigation results for the development feasibility of an HTS screening system which is intended to target MIG 12 and OIP5 genes, using a 96-well plate.
  • Example 1 Effects of MIG 12 and OIP5 overexpression on cells
  • Gateway Cloning Technology was chosen for the rapid cloning of human cDNA into the
  • S. pombe expression vector For the overexpression of human cDNA in S. pombe, the destination vector, pDES173 (KCTC 10757BP), was constructed by inserting a conversion cassette into pSLF173, an S. pombe expression vector containing an nmtl inducible promoter with an HA tag at the 5 '-end of the cloning site (http://www.atcc.org/ common/products/ vectors.cfm). The gene under the nmtl promoter is expressed in the absence of thiamine and subject to transcriptional repression in the presence of thiamine in S. pombe.
  • the genes of MIG12 and OIP5 human cDNAs in the pDONR201 donor vector 21C Frontier Human cDNA
  • ED665 Human genes in the pDES173 expression vector were introduced into S. pombe haploid strain ED665 (h-, ade6-M210 leul-32 ura4-D18) (Fanters, University of Glasgow, UK) by the method of lithium acetate. ED665 was cultured in Edinburgh Minimal Medium (EMM,
  • the phenotypic analysis was performed based on the hypothesis that the overexpression of genes involved in carcinogenesis induced growth inhibition and morphological changes.
  • S. pombe containing MIG 12 or OIP5 genes were washed 3 times, transferred to fresh media with or without thiamine, and grown for 8 hours.
  • S. pombe containing MIG 12 or OIP5 genes were analyzed for growth inhibition and morphological changes.
  • the expression of human genes MIGl 2 and OIP5 induced abnormal multi-septa formation of S. pombe, mostly caused by defects in the mitotic checkpoint and cytokinesis (FIGS. 2B and 3B).
  • human cDNAs were cloned into pDES173 under the nmtl promoter to be expressed as HA-tagged proteins at the N-terminal region in the absence of thiamine.
  • the overexpression of the human genes was clearly confirmed by Western blot analysis using an HA antibody (FIGS. 2D and 3D).
  • the phenotypic analysis indicated that the overexpression of MIGl 2 and OIP5 genes induced growth inhibition and morphological changes of S. pombe. Therefore, MIG 12 and OIP5 genes can be used as potential diagnostic markers and therapeutic targets of cancer.
  • the simple validation of a gene for relevance to cancer is to examine expression level of mRNA of the gene in cancer tissues or cancer cell lines. Comparison of mRNA levels between cancer and normal tissues provides clues that a gene of interest is involved in progression and regulation of carcinogenesis.
  • oncogenic validation was performed by examining the mRNA levels of the genes in various cancer cell lines.
  • the mRNA levels of MIG 12 and OIP5 genes in the various cancer cell lines can be measured by RT-PCR.
  • the stomach cancer cell lines used are Sun620, Sun216, Sun484, Sun ⁇ Ol and Sun638 (Korean Cell Line Bank, Seoul National University, Korea).
  • the liver cancer cell lines used are Huh7, SH-Jl, and Ck-Ia (by courtesy of Dr.
  • RT-PCR was performed with an RNA template (1 ⁇ g/reaction) and specific primers (Table 1) by using the One-step RT-PCR PreMix kit (iNtRON Biotechnology, Inc., Korea). PCR was carried out according to the instructions of the manufacturer: one cycle (45 0 C for 30 min, 94 ° C for 5 min), 30 cycles (94 ° C for 30 sec, 52 ° C for 30 sec, 72 ° C for 1 min), and one final extension cycle (72 "C for 5 min).
  • the genes of MIG 12 and OIP5 were highly expressed in most liver and stomach cancer cell lines compared to those in a fibroblast cell line, IMR90. OIP5 exhibited much higher expressions in stomach cancer cell lines than liver cancer cell lines (FIG. 4). These results indicate that the genes expressing at a high level in most of the cell lines may be associated with a common mechanism of liver and stomach cancers.
  • Example 3 3-1 Microarray analysis of QIP 5 in colorectal cancer
  • RNA samples were obtained from 66 patients of stage II and stage III at Samsung Medical Center (Seoul, Korea).
  • Total RNAs were extracted from cancer and normal tissues, using the Qiagen kit as described in the manual and used for microarray analysis of 48K Illumina chips. The quality of total RNA samples was assessed using an Experion (Bio- Rad Laboratories). RNA samples were labeled according to the chip manufacturers recommended protocols. 0.5 ⁇ g of total RNA from each sample was labeled by using the Illumina TotalPrep RNA Amplification Kit (Ambion) in a process of cDNA synthesis and in vitro transcription. Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-NTP (Ambion).
  • biotin-labeled cRNA was hybridized at 58 ° C for 16 hours to Illumina's Sentrix Human-6 v2 Expression BeadChip (Illumina).
  • the hybridized biotinylated cRNA was detected with streptavidin-Cy3 and quantitated using Illumina's BeadArray Reader Scanner (Illumina) according to manufacturer's instructions.
  • Microarray data of a total of 66 tumors and nine contiguous normal mucosa samples were processed and analyzed by Illumina BeadStudio version 3.0 software (Illumina). Data normalization was performed using quantile normalization, and fold changes and statistical significance were determined using the Avadis Prophetic version 3.3 (Strand Genomics). Scanned data was normalized by a quantile-quantile normalization method and log- transformed by base two. T-test was used to infer the significance of gene expression difference between tumor and normal tissues. Java Treeview (http://jtreeview.sourceforge.net/) was used to visualize the pattern of gene expression in tumors compared to normal tissues.
  • the up-regulating genes and down-regulating genes were shown in red and green respectively.
  • GAPDH Globallyceraldehyde 3 -phosphate dehydrogenase
  • ACTB Homo sapiens actin, beta
  • the expression profile of OIP5 in colorectal cancer tissues was examined and its expression level in tumor was compared with that of normal tissue (FIG. 5).
  • OIP5 was significantly up-regulated about 3.7-fold in colon tumors.
  • OIP5 was up-regulated over two fold in 58 out of 66 tumors.
  • no difference was noticed.
  • the expression levels of OIP5 among tumors of reoccurred and non-reoccurred patients were not very different.
  • OIP5 expression analysis of OIP5 in colorectal cancer tissues by RT-PCR
  • the mRNA levels of OIP5 in 10 cancer tissues randomly chosen among 66 colorectal cancer tissues were examined by reverse transcription-polymerase chain reaction (RT-PCR) (FIG. 6).
  • the OIP5 mRNA levels were higher in 80% (8/10) colorectal cancer tissues compared to normal tissue. This result was consistent with that of microarray analysis.
  • OIP5 expressed at a high level in most stomach and liver cancer cell lines tested suggests that OIP5 may be associated with a common mechanism of carcinogenesis of various cancers and can be used in diagnostics and therapeutics of cancer and screening of anticancer drugs.
  • Example 4 Increase of proliferation rate of cells by overexpression of OIP5 and MIG 12
  • Oncogenic genes can increase the proliferation rate of cells.
  • the proliferation rates of NIH3T3 cells transfected with MIG12 and OIP5 genes for 72 hours were examined as an indication of their oncogenic properties (FIG. 4).
  • NIH3T3 cells grown in 60 to 70% confluency in a 60 mm dish were transfected with 7 ⁇ g of DNA and 15 ⁇ i of LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, U. S. A) for 24 hours at 37 ° C in 5% CO 2 . Cells were harvested and counted using a hemacytometer.
  • the transient expression of the MIG 12 and OIP5 genes resulted in a two-fold increase in the proliferation rate (FIG. 7).
  • the increase of the proliferation rate by transient expression of a gene was coincident with foci formation and colony-forming assay.
  • RNA interference in which a sequence-specific gene-silencing process is mediated by small interfering RNA (siRNA), is an efficient method of identifying targets for gene therapy in a variety of diseases. Since OIP5 is highly expressed in cancer tissues, which is related to carcinogenesis, a decrease of OIP5 level may induce growth inhibition of cancer cells. To validate MIG 12 and OIP5 as a target in cancer therapeutics, RNAi experiments using siRNA in various cancer cells including colorectal cancer, liver cancer, stomach cancer, colorectal cancer and lung cancer were organized.
  • RNA oligonucleotides were custom- synthesized by Samchully Pharm. Co., Ltd. (Seoul, Korea) with an overhang of two thymidine residues (dTdT) at the 3' end.
  • RNA oligonucleotides were dissolved in Tris-EDTA (10 mM Tris-HCl, pH 8.0; and 1 rnM EDTA) as 200 ⁇ M solutions and were stored at -20 ° C.
  • Double-stranded siRNA molecules were generated by mixing the corresponding pair of sense and antisense RNA oligonucleotides in annealing buffer (30 mM HEPES-KOH, pH 7.9; 100 mM potassium acetate; and 2 mM magnesium acetate) at 20 ° C and then by incubating the reaction mixture at 95 " C for 2 min, followed by gradually cooling to room temperature.
  • the siRNAs were then aliquoted and stored at -20 "C .
  • siRNA target regions For MIG12 and OIP5, we calculated a relative binding energy pattern of candidate siRNAs so that cellular RISC (RNA-induced silencing complex) may bind and unwind siRNA double helix with ease. As shown in FIG. 8, the target regions were determined by the relative binding energy pattern search algorithm and siRNAs in the regions were designed and applied to our experiments. The relative binding energy pattern search algorithm determined the MIG 12
  • NM_011242 siRNA regions as those comprising SEQ ID NOS: 14, 15, 16, 17, and 18 extending contiguous 19-30 sequences and their complementaries. Sequences of SEQ ID NOS: 14, 15, 16, 17, and 18 are parts of MIG12 (NM_011242) sequences having both sense and complementary antisense strands in the range of nucleotides 845-950, 970-1030, 1080- 1160, 1220-1290, and 1320-1400, respectively. In other words, the regions for MIG12 candidate siRNAs include sequences of SEQ ID NOS: 14, 15, 16, 17, and 18 and the sequences comprising 19-30 contiguous nucleotide sequences and their complementaries.
  • the relative binding energy pattern search algorithm determined the OIP 5 (NM_021242) siRNA regions as those comprising SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25 and 26 extending contiguous 19-30 sequences and their complementaries. Sequences of SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25 and 26 are parts of OIP5 (NM_021242) sequences having both sense and complementary antisense strands in the range of nucleotides 1-60, 120-180, 380-460, 518-580, 720-780, 850-890, 920-980 and 1040-1100, respectively.
  • the regions for OIP5 candidate siRNAs include sequences of SEQ ID NOS: 19, 20, 21, 22, 23, 24, 25 and 26 and the sequences comprising 19-30 contiguous nucleotide sequences and their complementaries.
  • siMIG12 was designed to have a sequence of SEQ ID NO: 3 or 4 and siOIP5 was designed to have a sequence of SEQ ID NO: 5 or 6.
  • Sequences of SEQ ID NO: 3 and 4 have 1331 st to 1349 th nucleotides and 1123 rd to 1141 st nucleotides, respectively, of MIG 12 mRNA with two overhang nucleotides of TT.
  • Sequences of SEQ ID NO: 5 and 6 have 426 th to 444 th nucleotides and 417 th to 435 th nucleotides, respectively, of OIP5 mRNA with two overhang nucleotides of TT.
  • the cancer-related gene named RABGTTA (Rab geranylgeranyl transferase alpha- subunit gene), was targeted using siRNAs that have sequences of SEQ ID NOS: 7 and 8.
  • GFP Green Fluorescent Protein
  • AU siRNAs for the targeted genes were introduced into cells by synthesizing both sense and anti-sense strands followed by combining two in order to have double helix ribonucleotide bond.
  • siRNAs designed were synthesized by Samchully Pharm. Co., Ltd. (Seoul, Korea).
  • Example 6 Growth inhibition of cancer cells by RNAi of MIG 12 or OIP5 genes
  • various cancer cells including colorectal cancer, liver cancer, stomach cancer, and lung cancer were treated with siOIP5-l and siOIP5-2 and growth inhibition of cells was determined using SRB assay, flow cytometry analysis and mRNA measurements.
  • the knockdown of OIP5 was confirmed by RT- PCR and growth inhibition of cells was observed under a microscope.
  • the cancer cell lines used are Sun638 (stomach cancer, Korean Cell Line Bank, Seoul
  • HCC liver cancer, Korean Cell Line Bank, Seoul National University, Korea
  • Huh7 liver cancer, by courtesy of Dr. Dae Ghon Kim, Chonnam National
  • A549 lung cancer, Korean Cell Line Bank, Seoul National University, Korea
  • colo205 colonrectal cancer, Korean Cell Line Bank, Seoul National University, Korea
  • sw620 sw620
  • siRNA transfection Twenty-four hours before siRNA transfection, 1 x 10 5 cells were seeded in 6-well plates in RPMI-minimal essential medium (OptiMEM; Invitrogen, Grand Island, NY, USA) containing 10% Fetal Bovine Serum (FBS) with no antibiotics. Cells were seeded per 6-well plate to give 30% confluency at the time of the transfection.
  • the siMIG12 SEQ ID NOS: 3 and 4
  • siOIP5 SEQ ID NOS: 5 and 6
  • siRABGGTA SEQ ID NOS: 7 and 8
  • siGFP SEQ ID NO: 9
  • the cells received siGFP sequence were used as a control. After incubating the cells transfected with siRNA for 72 hours, SRB staining and microscopic observations were carried out (FIGS. 9 and 10). The knockdown of OIP5 was confirmed by RT-PCR and growth inhibition of cells was observed under a microscope.
  • siMIG12 showed strong effects on Hur7, SNU387, SNU638, colo205 and KM12C
  • siOIP5 showed strong effects on SNU387, SNU638, A549 and KM12C, thus suggesting cancer cell specific-RNAi effects of siMIG12 and siOIP5.
  • Introduction of both siMIG12 and siOIP5 into SNU638 (stomach cancer) caused growth inhibition (FIG. 10) and induced apoptosis as shown in FIG. 11.
  • CK-Ia liver cancer
  • the knockdown of RABGTTA a subunit of a Rab geranylgeranyl transferase, did not cause growth inhibition of cancer cells.
  • Apoptosis is an essential physiological process for the selective elimination of cells, which is involved in a variety of biological events.
  • RT-PCR of the genes involved in apoptosis was performed to examine their changes in mRNA levels during RNAi of MIG 12 or OIP5 (FIG. 12).
  • the expression level of Granzyme B increased when cells were treated with siOIP5.
  • Granzyme B a serine protease of cytotoxic T-lymphocytes (CTL) and NK cells, is able to directly activate caspases 3, 7, 8 and 10. Also, mRNAs of caspases 8, 9, 2, 5, 10, 4 were increased. They are pro-apoptotic species proteins.
  • Example 7 Construction of plasmid containing shMIG12 and shOIP5 To observe the RNAi effect by shMIG12 or shOIP5, a plasmid containing shRNA was constructed (FIG. 13A). After transfection of cells with the plasmid containing shRNA, growth inhibition of cancer cells was examined. The shRNA sequences of top and down complementary strands were designed. Each strand (67 mer/each) consisting of a restriction enzyme site, a target sense siRNA sequence, a loop sequence and a nonsense siRNA sequence, which is gatcc-siRNA sense-loop-siRNA reverse-TTTTTTACGCGTg, was designed and synthesized.
  • Sequences of SEQ ID NOS: 27 and 28 represent top shRNA strands of siMIG12 containing a sequence of SEQ ID NO: 4 and siOIP5 containing a sequence of SEQ ID NO: 5 respectively.
  • the sequence of SEQ ID NO: 29 represents a top shRNA strand of siGFP containing a sequence of SEQ ID NO: 9.
  • the annealed oligonucleotides were cloned into pENTR-U6-BHRNX (Neurogenex) and used for RNAi experiments as shown in FIG. 13 A.
  • Example 8 Inhibition of cancer cell growth by shMIG12 or shOIP5
  • Huh7 liver cancer cell line
  • Huh7 liver cancer cell line
  • SRB staining The cells transfected with shMIG12 or shOIP5 showed growth inhibition, while the cells with shGFP were not affected, thus indicating that shMIG12 or shOIP5 can be developed as cancer therapeutics.
  • Example 9 Application of S. pornbe cells overexpressing MIG12 or QIPS When MIG 12 or OIP5 was overexpressed in S. pombe, cell growth was inhibited, suggesting that MIG 12 and OIP5 might be involved in cell signaling.
  • MIGl 2 or OIP5 under the nmtl promoter is subject to transcriptional repression in the presence of thiamine (EMM+AL+Thia) and overexpressed in the absence of thiamine (EMM+AL) in S. pombe. Therefore, S. pombe overexpressing MIGl 2 or OIP5 in the absence of thiamine can be exploited to identify compounds or molecules relieving growth inhibition of cells using a chemical library.
  • FIG. 15A and spotted in the plate (FIG. 15B).
  • EMM+AL thiamine
  • MIG 12 and OIP5 genes of the present invention are expressed at relatively high levels in cancer cell lines and increase a proliferation rate of normal cells. Therefore, inhibition of expression of these genes via RNAi using siRNA or shRNA leads to suppression of cancer cell growth. For these reasons, MIG 12 and OIP5 genes can be used as a target gene for diagnosis or cancer therapy of various cancers. As a result, it is expected that the present invention will significantly contribute to the development of biopharmaceuticals and synthetic/natural medicines which are aimed at development of anticancer drugs having low adverse side effects, such as cancer target gene-specific therapeutic agents. Further, the present invention will contribute to the development of a source technology which forms the basis of study of new cancer-related mechanisms.

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Abstract

La présente invention concerne une composition pour le diagnostic du cancer ou le criblage de medicaments anti-cancéreux comportant un gène MIG12 ou une proteine exprimée par le gène MIG12, une composition pour le diagnostic du cancer ou le criblage de medicaments anti-cancéreux comportant un gène OIP5 12 ou une proteine exprimée par le gène PIO5, une composition comprenant un inhibiteur desdits gènes ou un inhibiteur desdites protéines et un support pharmaceutiquement acceptable, et une trousse de diagnostic du cancer comportant au moins un parmi un gène MIG12, un gène OIP5, et une protéine exprimée par le gène MIG12 ou le gène PIO5. Les gènes MIG12 et PIO5 selon la présente invention sont exprimés à des niveaux relativement élevés dans des lignées cellulaires cancéreuses et accroissent un taux de prolifération de cellules normales. Par conséquent, l'inhibition de l'expression de ces gènes via l'ARNi par l'ARNsi ou l'ARNsh entraîne la suppression de croissance de cellules cancéreuses. Pour toutes ces raisons, les gènes MIG12 et OIP5 peuvent être utilisés comme une cible pour le diagnostic ou la thérapie du cancer de divers cancers.
PCT/KR2007/006366 2006-12-08 2007-12-07 Nouvelle utilisation de gènes mig12 et pio5 Ceased WO2008069621A1 (fr)

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WO2010023864A1 (fr) * 2008-08-28 2010-03-04 Oncotherapy Science, Inc. Oip5 en tant que gène cible pour le traitement et le diagnostic du cancer
WO2010099341A1 (fr) * 2009-02-26 2010-09-02 Alnylam Pharmaceuticals, Inc. Composition et procédés d'inhibition de l'expression du gène mig-12

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US20040171037A1 (en) * 2002-11-19 2004-09-02 Jing Li Amplified genes involved in cancer
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010023864A1 (fr) * 2008-08-28 2010-03-04 Oncotherapy Science, Inc. Oip5 en tant que gène cible pour le traitement et le diagnostic du cancer
WO2010099341A1 (fr) * 2009-02-26 2010-09-02 Alnylam Pharmaceuticals, Inc. Composition et procédés d'inhibition de l'expression du gène mig-12
US20120041051A1 (en) * 2009-02-26 2012-02-16 Kevin Fitzgerald Compositions And Methods For Inhibiting Expression Of MIG-12 Gene

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