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WO2022044788A1 - Double-stranded nucleic acid molecule, dna, vector, female cancer cell growth inhibition agent, female cancer tumor formation inhibition agent, pharmaceutical, and use of long-chain noncoding rna - Google Patents

Double-stranded nucleic acid molecule, dna, vector, female cancer cell growth inhibition agent, female cancer tumor formation inhibition agent, pharmaceutical, and use of long-chain noncoding rna Download PDF

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WO2022044788A1
WO2022044788A1 PCT/JP2021/029473 JP2021029473W WO2022044788A1 WO 2022044788 A1 WO2022044788 A1 WO 2022044788A1 JP 2021029473 W JP2021029473 W JP 2021029473W WO 2022044788 A1 WO2022044788 A1 WO 2022044788A1
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cancer
double
female
female cancer
nucleic acid
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Japanese (ja)
Inventor
聡 井上
和博 池田
公仁子 井上
俊彦 竹岩
悠一 水戸部
幸清 長谷川
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Saitama Medical University
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Saitama Medical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention is a double-stranded nucleic acid molecule that can be suitably used for the prevention or treatment of female cancer, a DNA containing a sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, and the double-stranded nucleic acid molecule. , At least one of the female cancer cell growth inhibitor or the female cancer tumor formation inhibitor, the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor, which contains at least one of the DNA and the vector.
  • the present invention relates to a drug containing any of these, a method for evaluating female cancer using the presence or absence of a long-stranded non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 or the expression level thereof as an index, and a marker for evaluating female cancer.
  • Ovarian cancer which is one of the female cancers, is one of the most prevalent cancers in women (see Non-Patent Document 1).
  • GLOBOCAN2018 created by the International Agency for Research on Cancer (IARC), which is an external organization of the World Health Organization
  • IARC International Agency for Research on Cancer
  • ovarian cancer affects all cancer types in women worldwide. It is the eighth highest among them, and the number of new cases and deaths is estimated to be 295,414 and 184,779, respectively (see Non-Patent Document 2).
  • treatments for ovarian cancer continue to advance, ovarian cancer mortality remains the highest among female cancers (see Non-Patent Document 3).
  • Treatment tends to be successful for early-detected ovarian cancer, but early-stage ovarian cancer is less symptomatic and is diagnosed only when about 60% of cases are more advanced. This contributes to the 5-year survival rate of patients with ovarian cancer falling below 50% (see Non-Patent Documents 4 to 6). Therefore, countermeasures for ovarian cancer are an urgent issue, and new biomarkers, therapeutic targets, etc. are indispensable for improving the treatment results of ovarian cancer.
  • Non-coding RNAs are classified by length, and in particular non-coding RNAs longer than 200 bases are defined as long noncoding RNAs (lncRNAs). It is said that a huge number of long-chain non-coding RNA genes exist in the human genome. For example, NONCODE (http://http: //), which is a database of non-coding RNA expressed in 17 kinds of organisms including human and mouse. www.noncode.org/) has registered as many as 96,308 human long non-coding RNA genes. Although most of the long non-coding RNAs are still unclear in function, some long non-coding RNAs have been shown to play an important role in the pathophysiology of cancer (Non-Patent Documents 7-10). reference). However, research on long-chain non-coding RNAs is still young, and the function of most long-chain non-coding RNAs remains unknown, and is clinically applied as a marker or therapeutic target in female cancers such as ovarian cancer. There is nothing that is.
  • the present invention is a double-stranded nucleic acid molecule that can effectively suppress the growth of female cancer cells and tumorigenesis of female cancer and can be suitably used for the prevention or treatment of female cancer.
  • a female cancer cell growth inhibitor or a female cancer containing at least one of a DNA containing a base sequence encoding a double-stranded nucleic acid molecule, a vector containing the DNA, the double-stranded nucleic acid molecule, the DNA, and the vector.
  • RNA 1 hereinafter sometimes referred to as“ OIN1 ”
  • OIN1 RNA 1
  • a double-stranded nucleic acid molecule that efficiently suppresses the expression of the long-stranded non-coding RNA and introducing the double-stranded nucleic acid molecule into a cancer cell or a tumor, the growth of the cancer cell and the above-mentioned It was found that tumor formation was suppressed. Furthermore, it has been found that in cancer cells into which the double-stranded nucleic acid molecule has been introduced, apoptosis is induced by a decrease in the expression of the long-chain non-coding RNA.
  • ⁇ 1> A double-stranded nucleic acid molecule for suppressing the expression of a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
  • B A double-stranded nucleic acid molecule comprising the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
  • the DNA is characterized by containing a base sequence encoding the double-stranded nucleic acid molecule described in ⁇ 1>.
  • ⁇ 3> A vector comprising the DNA described in ⁇ 2> above.
  • Female cancer cell proliferation comprising at least one of the double-stranded nucleic acid molecule described in ⁇ 1>, the DNA described in ⁇ 2>, and the vector described in ⁇ 3>. It is an inhibitor.
  • ⁇ 5> A method for suppressing the growth of female cancer cells, which comprises allowing the female cancer cell growth inhibitor according to ⁇ 4> to act on the female cancer cells.
  • ⁇ 6> A tumor of female cancer characterized by containing at least one of the double-stranded nucleic acid molecule described in ⁇ 1>, the DNA described in ⁇ 2>, and the vector described in ⁇ 3>. It is a formation inhibitor.
  • ⁇ 7> A method for suppressing tumor formation of female cancer, which comprises allowing the tumor formation inhibitor for female cancer according to ⁇ 6> to act on a tumor of female cancer.
  • ⁇ 8> A drug for preventing or treating female cancer, which is at least one of the female cancer cell growth inhibitor according to ⁇ 4> and the tumor formation inhibitor for female cancer according to ⁇ 6>. It is a medicine characterized by containing any of them.
  • ⁇ 9> A method for preventing or treating female cancer, which comprises administering the drug according to the above ⁇ 8> to an individual.
  • it is a method for evaluating female cancer which comprises evaluating whether or not the patient has a possibility of suffering from female cancer.
  • a marker for evaluation of female cancer which comprises a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
  • a drug containing at least one of a female cancer cell growth inhibitor or a female cancer tumor formation inhibitor, the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor, a method for evaluating female cancer, And markers for evaluation of female cancer can be provided.
  • FIG. 1A is a diagram showing the results of comparing the expression level of OIN1 in the normal tissue of the ovary and the specimens of clear cell ovarian cancer and highly atypical serous ovarian cancer.
  • FIG. 1B is a diagram showing the results of mapping RNA sequencing leads derived from normal ovarian tissue and specimens of clear ovarian cell cancer and highly atypical serous ovarian cancer to the OIN1 locus.
  • FIG. 1C is a diagram showing the results of examining the expression of OIN1 in ovarian cancer cells by the qRT-PCR method.
  • FIG. 2A is a diagram showing the results of analysis of the knockdown efficiency of OIN1 by siRNA with respect to OIN1 by qRT-PCR with A2780 (left), SKOV3 (center), and RMG1 (right).
  • FIG. 2B is a diagram showing the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of A2780 (left), SKOV3 (center), and RMG1 (right).
  • FIG. 2C is FIG. 1 showing the results of analysis of apoptosis using A2780 cells.
  • FIG. 2D is FIG. 1 showing the results of analysis of apoptosis using SKOV3 cells.
  • FIG. 2E is FIG. 2 showing the results of analysis of apoptosis using A2780 cells.
  • FIG. 1 is a diagram showing the results of analysis of the knockdown efficiency of OIN1 by siRNA with respect to OIN1 by qRT-PCR with A2780 (left), SKOV3 (center), and RMG1 (right
  • FIG. 2F is FIG. 2 showing the results of analysis of apoptosis using SKOV3 cells.
  • FIG. 3A is a diagram showing the results of analysis regarding the effect of knockdown of OIN1 on the expression of various genes.
  • FIG. 3B is FIG. 2 showing the results of analysis regarding the effect of knockdown of OIN1 on the expression of various genes.
  • FIG. 3C is a diagram showing the results of analysis of the expression patterns of the RASSF5 gene (upper figure) and the ADORA1 gene (lower figure) from RNA-Seqing data in a clinical specimen of highly atypical serous ovarian cancer.
  • FIG. 4A is a diagram showing an example of an ovarian cancer xenograft tumor model mouse injected with each siRNA.
  • FIG. 4A is a diagram showing an example of an ovarian cancer xenograft tumor model mouse injected with each siRNA.
  • FIG. 4B is a diagram showing the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA.
  • FIG. 4C is a diagram showing the results of excising a xenograft tumor and measuring the weight.
  • FIG. 4D is a diagram showing the results of examining the expression of OIN1 in A2780-derived xenograft tumors injected with each siRNA.
  • FIG. 4E is a diagram showing the results of examining the expression of RASSF5 in A2780-derived xenograft tumors injected with each siRNA.
  • FIG. 4F is a diagram showing the results of examining the expression of ADORA1 in A2780-derived xenograft tumors injected with each siRNA.
  • FIG. 5A is a diagram showing the results of processing shiOIN1 # 1 or siOIN1 # 2 on Ishikawa and analyzing the knockdown efficiency of OIN1 by qRT-PCR.
  • FIG. 5B is a diagram showing the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of Ishikawa.
  • FIG. 5C is a diagram showing the results of processing siOIN1 # 1 on BrC-PDC and analyzing the knockdown efficiency of OIN1 by qRT-PCR.
  • FIG. 5D is a diagram showing the results of investigation by CellTiter-3D Cell Viability Assay (Promega) that knockdown of OIN1 suppresses the proliferation of BrC-PDC.
  • FIG. 6A is a diagram showing the results of analysis of proliferation of A2780 cells transfected with the OIN1 expression plasmid or an empty vector using a DNA assay.
  • FIG. 6B is a diagram showing the results of analysis of proliferation of SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector using a DNA assay.
  • FIG. 6C is a diagram showing the results of confirming changes in the expression of OIN1 RNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 6D is a diagram showing the results of confirming changes in the expression of RASSF5 mRNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 6E is a diagram showing the results of confirming changes in the expression of ADORA1 mRNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 6F is a diagram showing the results of confirming changes in the expression of OIN1 RNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 6G is a diagram showing the results of confirming changes in the expression of RASSF5 mRNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 6H is a diagram showing the results of confirming changes in the expression of ADORA1 mRNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector.
  • FIG. 7 is a diagram showing the results of comparing the expression level of OIN1 in a normal tissue of an ovary and a sample of endometrial cancer.
  • the double-stranded nucleic acid molecule of the present invention is a double-stranded nucleic acid molecule for suppressing the expression of a long-chain non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1; (a) SEQ ID NO: 2 And the sense strand containing the base sequence corresponding to the target sequence consisting of the base sequence represented by any of SEQ ID NO: 3 and (b) the sense strand forming a double strand with the sense strand of (a) above. Includes an antisense strand containing a complementary base sequence.
  • the "double-stranded nucleic acid molecule” refers to a double-stranded nucleic acid molecule formed by hybridizing a sense strand and an antisense strand.
  • RNA consisting of a base sequence represented by SEQ ID NO: 1 The long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is referred to as NONHSAT103448 in the NONCODE database (NONCODE. Available on line: http: //www.noncode.org/ (accessed on 31 May 2020).). It was registered, but no research has been done so far, and its function is unknown. Therefore, the present inventors have named a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 as ovarian cancer long intergenic noncoding RNA 1 (OIN1). As shown in the test examples described later, OIN1 is overexpressed in female cancer cells and has a function of suppressing apoptosis of female cancer cells and promoting proliferation of female cancer cells.
  • the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is targeted by the double-stranded nucleic acid molecule, and its expression is suppressed by the double-stranded nucleic acid molecule.
  • a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 may be referred to as a "target RNA" of the double-stranded nucleic acid molecule.
  • the double-stranded nucleic acid molecule of the present invention includes (a) a sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3 and (b). ) It includes the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
  • the sense strand and the antisense strand may be an RNA strand or an RNA-DNA chimeric strand.
  • the sense strand and the antisense strand can hybridize with each other to form the double-stranded nucleic acid molecule.
  • the sense strand in the double-stranded nucleic acid molecule may contain a base sequence corresponding to the target sequence, may contain other base sequences, and consists only of the base sequence corresponding to the target sequence. It may be a thing.
  • the antisense strand in the double-stranded nucleic acid molecule may contain a base sequence complementary to the extent that it can hybridize with the sense strand, and may contain other base sequences.
  • the base sequence complementary to the sense strand is preferably contained in an amount of 70% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.
  • the type of the double-stranded nucleic acid molecule is not particularly limited and may be appropriately selected depending on the intended purpose.
  • double-stranded RNA dsRNA
  • double-stranded RNA-DNA chimera etc.
  • double-stranded RNA refers to a double-stranded nucleic acid molecule in which both the sense strand and the antisense strand are composed of RNA sequences
  • double-stranded RNA-DNA chimera refers to sense.
  • a double-stranded nucleic acid molecule in which both the strand and the antisense strand are composed of a chimeric sequence of RNA and DNA.
  • the double-stranded RNA and double-stranded RNA-DNA chimera are preferably siRNA (small interfering RNA) or chimeric siRNA, and more preferably siRNA.
  • the siRNA is a small molecule double-stranded RNA having a length of 18 bases to 29 bases, and the target RNA having a sequence complementary to the antisense strand (guide strand) of the siRNA is cleaved to obtain the target RNA. It has a function of suppressing expression.
  • the terminal structure thereof is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the siRNA may have a blunt end or a protruding end (overhang).
  • the siRNA preferably has a structure in which the 3'end of each strand protrudes by 2 to 6 bases, and more preferably has a structure in which the 3'end of each strand protrudes by 2 bases.
  • the chimeric siRNA refers to a small molecule double-stranded RNA-DNA chimera having a length of 18 bases to 29 bases in which a part of the RNA sequence of siRNA is converted into DNA.
  • a small molecule double strand with a length of 21 to 23 bases in which the bases within 8 bases on the 3'side of the sense strand of siRNA and within 6 bases on the 5'side of the antisense strand are converted into DNA. It is preferably an RNA-DNA chimera.
  • the chimeric siRNA has a function of suppressing the expression of a target gene, similarly to the siRNA.
  • the chimeric siRNA also includes an embodiment in which a part of the sequence converted into DNA is converted into RNA again.
  • the terminal structure of the chimeric siRNA is not particularly limited as in the case of the siRNA, and can be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang). It may have.
  • siRNA examples include the following.
  • siRNA whose target sequence is the base sequence represented by the SEQ ID NO: 2
  • examples of the siRNA whose target sequence is the base sequence represented by the SEQ ID NO: 2 include siRNA composed of the following sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 5.
  • siRNA composed of the following sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 5.
  • siRNA in which the target sequence is the base sequence represented by the SEQ ID NO: 3 examples include the siRNA composed of the following sense strand of SEQ ID NO: 6 and the antisense strand of SEQ ID NO: 7. Be done. -Sense strand 5'-GACAGGAGACUCCAGAAAGG-3'(SEQ ID NO: 6) -Antisense strand 5'-UUUUCUGGAGUCUCCUGUCUG-3'(SEQ ID NO: 7)
  • the double-stranded RNA may be shRNA (short hairpin RNA).
  • shRNA short hairpin RNA
  • the shRNA is a single-stranded RNA containing a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases, but the shRNA is a base pair when expressed in vivo. Is formed into a hairpin-shaped double-stranded RNA.
  • the shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to suppress the expression of the target RNA.
  • Dicer RNase III enzyme
  • the terminal structure of the shRNA is not particularly limited as in the siRNA and the double-stranded RNA-DNA chimera, and can be appropriately selected depending on the intended purpose. For example, it may have a blunt end. It may have a protruding end (overhang).
  • the double-stranded nucleic acid molecule may have appropriate modifications depending on the purpose.
  • the double-stranded nucleic acid molecule is modified with 2'O-methyl or phosphorothioated for the purpose of imparting resistance to a nucleic acid-degrading enzyme (nuclease) and improving stability in a culture solution or in a living body.
  • S conversion nucleic acid-degrading enzyme
  • LNA Locked Nucleic Acid
  • the 5'end or 3'end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide or the like. You can also.
  • the method for applying such a modification to the double-stranded nucleic acid molecule is not particularly limited, and a conventionally known method can be appropriately used.
  • the method for obtaining the double-stranded nucleic acid molecule is not particularly limited, and each can be produced based on a conventionally known method.
  • the siRNA chemically synthesizes a single-stranded RNA having a length of 18 to 29 bases corresponding to a desired sense strand and an antisense strand, respectively, using an existing automatic DNA / RNA synthesizer or the like. And can be made by annealing them.
  • a commercially available double-stranded siRNA that has been annealed can be obtained, or it can be obtained by requesting synthesis from a siRNA synthesis contractor.
  • siRNA can be produced by utilizing the intracellular reaction.
  • the chimeric siRNA can be produced, for example, by chemically synthesizing a sense strand and an antisense strand, which are chimeric nucleic acid molecules, and annealing them.
  • the DNA of the present invention is a DNA containing a base sequence encoding the double-stranded nucleic acid molecule of the present invention described above, and the vector of the present invention is a vector containing the DNA.
  • the DNA is not particularly limited as long as it is a DNA containing a base sequence encoding the double-stranded nucleic acid molecule of the present invention, and can be appropriately selected depending on the intended purpose. It is preferable that a promoter sequence for controlling transcription of the double-stranded nucleic acid molecule is linked upstream (5'side) of the encoding base sequence.
  • the promoter sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a pol II promoter such as a CMV promoter, a pol III promoter such as an H1 promoter and a U6 promoter, and the like.
  • the terminator sequence for terminating the transcription of the double-stranded nucleic acid molecule is linked downstream (3'side) of the base sequence encoding the double-stranded nucleic acid molecule.
  • the terminator sequence is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a transcription unit comprising the promoter sequence, the base sequence encoding the double-stranded nucleic acid molecule, and the terminator sequence is a preferred embodiment in the DNA.
  • the transfer unit can be constructed by using a conventionally known method.
  • the vector is not particularly limited as long as it contains the DNA, and can be appropriately selected depending on the intended purpose. Examples thereof include a plasmid vector and a virus vector.
  • the vector is preferably an expression vector capable of expressing the double-stranded nucleic acid molecule.
  • the expression mode of the double-stranded nucleic acid molecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, as a method for expressing siRNA as a double-stranded nucleic acid molecule, two short single-stranded RNAs are used. Examples thereof include a method for expressing (tandem type), a method for expressing a single-stranded RNA as shRNA (hairpin type), and the like.
  • the tandem-type siRNA expression vector contains a DNA sequence encoding a sense strand constituting the siRNA and a DNA sequence encoding an antisense strand, and is upstream (5'side) of the DNA sequence encoding each strand.
  • the promoter sequence is ligated to each, and the terminator sequence is ligated downstream (3'side) of the DNA sequence encoding each strand.
  • the DNA sequence encoding the sense strand constituting the siRNA and the DNA sequence encoding the antisense strand are arranged in opposite directions, and the sense strand DNA sequence and the antisense strand DNA are arranged in opposite directions.
  • sequences are connected via a loop sequence, and contain DNA to which the promoter sequence is linked upstream (5'side) and the terminator sequence is linked downstream (3'side).
  • Each of the vectors can be constructed by using a conventionally known method, for example, by ligating the DNA to a cleavage site of a vector previously cut with a restriction enzyme.
  • the promoter By introducing (transfecting) the DNA or the vector into a cell, the promoter can be activated and the double-stranded nucleic acid molecule can be produced.
  • the DNA is transcribed intracellularly to generate a sense strand and an antisense strand, and hybridizing them produces siRNA.
  • the DNA is transcribed intracellularly to first generate hairpin-type RNA (SHRNA), and then processing by a dicer produces siRNA.
  • SHRNA hairpin-type RNA
  • the female cancer cell growth inhibitor of the present invention is for suppressing the growth of female cancer cells, and contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, and further. Includes other ingredients as needed.
  • the tumor formation inhibitor for female cancer of the present invention is for suppressing the formation of a tumor for female cancer, and contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above. , And other ingredients as needed.
  • Double-stranded nucleic acid molecule DNA, vector>
  • the details of the double-stranded nucleic acid molecule are as described in the above-mentioned item of the double-stranded nucleic acid molecule of the present invention. Since the double-stranded nucleic acid molecule can effectively suppress the expression of long-chain non-coding RNA consisting of the base sequence represented by the target SEQ ID NO: 1, it suppresses the proliferation of female cancer cells. It is suitable as an active ingredient of the female cancer cell proliferation inhibitor for the purpose of, or as an active ingredient of the tumor formation inhibitor of the female cancer for suppressing the formation of a female cancer tumor.
  • the total content of at least one of the double-stranded nucleic acid molecule, DNA, and vector in the female cancer cell growth inhibitor or the tumor formation inhibitor of female cancer is not particularly limited, and may vary depending on the intended purpose. It can be selected as appropriate. Further, the female cancer cell growth inhibitor or the female cancer tumor formation inhibitor may be at least one of the double-stranded nucleic acid molecule, DNA, and vector itself.
  • the other components are not particularly limited and may be appropriately selected depending on the intended purpose.
  • physiology for diluting at least one of the double-stranded nucleic acid molecule, DNA, and vector to a desired concentration examples thereof include diluting agents such as saline solution and culture solution, and transfection reagents for introducing (transfecting) at least one of the double-stranded nucleic acid molecule, DNA, and vector into a target cell or tumor. Be done.
  • the content of the other components in the female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the female cancer to which the female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer is applied is not particularly limited and may be appropriately selected depending on the intended purpose, but ovarian cancer and uterus. At least one of cancer (including uterine body cancer and cervical cancer) and breast cancer is preferably mentioned. The types of ovarian cancer, uterine cancer, and breast cancer are not particularly limited and may be appropriately selected depending on the intended purpose.
  • the female cancer cell may be a cell cultured outside the body or a cell existing in the body of an individual.
  • the female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor can be introduced into, for example, a female cancer cell or a female cancer tumor to cause the female cancer cell or the female cancer tumor.
  • the method of introduction is not particularly limited and may be appropriately selected from conventionally known methods according to the purpose. For example, a method using a transfection reagent, a method using electroporation, or a method using magnetic particles. , A method using virus infection, a method of injecting by injection, etc.
  • the amount of the female cancer cell proliferation inhibitor or the tumor formation inhibitor of female cancer acting on the female cancer cell or the tumor of the female cancer is not particularly limited, and the type of cell or tumor may be used.
  • the amount of the active ingredient is preferably about 0.1 ⁇ g with respect to the number of cells of 1 ⁇ 10 6 .
  • About 5 ⁇ g is more preferable, and about 15 ⁇ g is particularly preferable.
  • the female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer of the present invention may be used alone, in combination with each other, or with other therapeutic agents for female cancer. It may be used in combination.
  • the present invention also relates to a method for suppressing the growth of female cancer cells, which comprises allowing the female cancer cell growth inhibitor to act on female cancer cells.
  • the tumor formation inhibitor for female cancer contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, it is represented by the above-mentioned SEQ ID NO: 1 by acting on the tumor of female cancer.
  • the formation of tumors of female cancer can be effectively suppressed through the suppression of the expression of long-chain non-coding RNA consisting of the base sequence. Therefore, the present invention also relates to a method for suppressing tumor formation in female cancer, which comprises allowing the tumor formation inhibitor for female cancer to act on a tumor in female cancer.
  • the female cancer is not particularly limited, and examples thereof include the same as those described in the item of ⁇ female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done. Further, in the method for suppressing the growth of female cancer cells or the method for suppressing tumor formation of female cancer, another female cancer therapeutic agent may be further acted upon.
  • the medicine of the present invention is a medicine for preventing or treating female cancer, and includes at least one of the above-mentioned female cancer cell growth inhibitor and female cancer tumor formation inhibitor of the present invention, and further. Contains other ingredients as needed.
  • the female cancer is not particularly limited, and examples thereof include the same as those described in the item of ⁇ female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.
  • the female cancer cell growth inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, the length consisting of the base sequence represented by the above-mentioned SEQ ID NO: 1 as a target.
  • the growth of female cancer cells can be effectively suppressed through the suppression of the expression of non-coding RNA. That is, the female cancer cell growth inhibitor can be suitably used as a medicine for preventing or treating female cancer.
  • the tumor formation inhibitor for female cancer contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, it comprises the base sequence represented by the above-mentioned SEQ ID NO: 1 as a target.
  • the tumor formation inhibitor for female cancer can be suitably used as a medicine for preventing or treating female cancer.
  • the total content of at least one of the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor in the drug is not particularly limited and may be appropriately selected depending on the intended purpose. Further, the drug may consist of at least one of the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor.
  • the double-stranded nucleic acid molecule that is the active ingredient of the pharmaceutical the double-stranded nucleic acid molecule itself in an unmodified state may be used, but it is applied to a living body so that an appropriate preventive or therapeutic effect can be obtained. It is preferable to use a double-stranded nucleic acid molecule in a form suitable for administration of.
  • the double-stranded nucleic acid molecule is preferably modified in that the stability of the double-stranded nucleic acid molecule in a living body can be enhanced.
  • the type of modification that can be applied to the double-stranded nucleic acid molecule is not particularly limited, and examples thereof include 2'O-methyl modification, phosphorothioate (S) modification, and LNA (Locked Nucleic Acid) modification.
  • the 5'end or 3'end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide or the like. It is also preferable.
  • the method for applying the modification to the double-stranded nucleic acid molecule is not particularly limited, and a conventionally known method can be appropriately used.
  • the double-stranded nucleic acid molecule forms a complex with a liposome, a polymer matrix, or the like in that the introduction efficiency into the target cell can be enhanced.
  • the method for forming the complex is not particularly limited, and a conventionally known method can be appropriately used.
  • the other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a pharmaceutically acceptable carrier.
  • the carrier is also not particularly limited and may be appropriately selected depending on, for example, a dosage form or the like. Further, the content of the other component in the medicine is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the dosage form of the pharmaceutical is not particularly limited and may be appropriately selected depending on the desired administration method, for example, an oral solid preparation (tablet, coated tablet, granule, powder, capsule, etc.). Oral solutions (oral solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solids for errands, etc.), ointments, patches, gels, creams, external powders, sprays, inhalations. Examples include powders.
  • an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / flavoring agent, etc. are added to the active ingredient, and a conventional method is used.
  • an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / flavoring agent, etc.
  • Can be manufactured by Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
  • binder examples include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Be done.
  • disintegrant examples include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, lactose and the like.
  • lubricant examples include purified talc, stearate, borax, polyethylene glycol and the like.
  • colorant examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.
  • the oral solution for example, it can be produced by a conventional method by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to the active ingredient.
  • additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer
  • the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.
  • the buffer include sodium citrate and the like.
  • the stabilizer include tragant, gum arabic, gelatin and the like.
  • a pH regulator, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the active ingredient, and the injection is subcutaneously, intramuscularly, or intravenously used by a conventional method. Etc. can be produced.
  • the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like.
  • the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid and the like.
  • the tonicity agent include sodium chloride, glucose and the like.
  • the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.
  • a known base, stabilizer, wetting agent, preservative and the like can be blended with the active ingredient and mixed by a conventional method to produce the ointment.
  • the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, paraffin and the like.
  • the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.
  • a cream, a gel, a paste, or the like as the ointment can be applied to a known support by a conventional method to produce the patch.
  • the support include cotton, rayon, woven fabric made of chemical fibers, non-woven fabric, soft vinyl chloride, polyethylene, polyurethane and other films, foam sheets and the like.
  • the drug is suitable for the prevention or treatment of female cancer. Therefore, the said medicine can be suitably used by administering it to an individual who has or may have suffered from female cancer.
  • the individual to be administered with the drug is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs and cats. Of these, humans are particularly preferred.
  • the method of administering the drug is not particularly limited, and for example, either local administration or systemic administration can be selected according to the dosage form of the drug, the type of disease, the condition of the patient, and the like.
  • the active ingredient of the drug (the double-stranded nucleic acid molecule) can be administered by directly injecting it into a desired site (for example, a tumor site).
  • a desired site for example, a tumor site
  • a conventionally known method such as injection can be appropriately used.
  • systemic administration for example, oral administration, intraperitoneal administration, administration into blood, etc.
  • the active ingredient of the drug is stably delivered to a desired site (for example, a tumor site). It is preferable to appropriately apply a conventionally known drug delivery technique so that the drug can be delivered efficiently.
  • the dose of the drug is not particularly limited and may be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the patient to be administered. For example, per daily administration to an adult.
  • the amount of the active ingredient (the double-stranded nucleic acid molecule) is preferably 1 mg to 100 mg.
  • the number of administrations of the drug is not particularly limited, and can be appropriately selected depending on the age, body weight, degree of desired effect, and the like of the patient to be administered.
  • the administration time of the drug is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be administered prophylactically or therapeutically for a disease. Above all, since the drug is excellent in the effect of inhibiting the growth of female cancer cells and the effect of suppressing the formation of tumors of female cancer, the drug should be administered at the earliest possible stage of the disease. Is considered desirable.
  • the pharmaceutical product of the present invention may be used in combination with other female cancer therapeutic agents.
  • the drug comprises at least one of the female cancer cell proliferation inhibitor and the female cancer tumor growth inhibitor, it is targeted by administration to an individual who has or may have suffered from female cancer. It effectively suppresses at least one of the proliferation of female cancer cells and the formation of female cancer tumors through the suppression of the expression of long-chain non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. , Can prevent or treat female cancer. Therefore, the present invention also relates to a method for preventing or treating female cancer, which comprises administering the above-mentioned medicine to an individual.
  • the female cancer is not particularly limited, and examples thereof include the same as those described in the item of ⁇ female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done. Further, in the preventive or therapeutic method, another female cancer therapeutic agent may be further administered.
  • the method for evaluating female cancer of the present invention includes at least an evaluation step, and if necessary, includes other steps such as a detection step.
  • the female cancer is not particularly limited, and examples thereof include the same as those described in the item of ⁇ female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.
  • ⁇ Evaluation process> whether or not the subject suffers from female cancer using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index. It is a step of evaluating whether or not a woman has a possibility of developing cancer.
  • sample derived from the subject is not particularly limited as long as it is prepared from a target individual, and can be appropriately selected according to the purpose. Examples thereof include cells, tissues, and blood at the lesion site. Be done.
  • the sample may be further subjected to RNA preparation treatment or the like. As the sample, only one kind may be used, or two or more kinds may be used.
  • the subject is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs and cats, and among these, humans. Is particularly preferable.
  • the long-chain non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is the long-chain non-coding RNA consisting of the base sequence represented by ⁇ SEQ ID NO: 1 of the above-mentioned (double-stranded nucleic acid molecule) of the present invention. As described in the item of RNA>.
  • the detection step is a step of detecting a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 in a sample prepared from an individual.
  • the detection method is not particularly limited, and a known method can be appropriately selected depending on the intended purpose. Examples thereof include a method by qRT-PCR and a method by RNA sequencing. These may be used alone or in combination of two or more.
  • the primer set used for the PCR is not particularly limited as long as it can specifically amplify the base sequence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. You can choose.
  • primer set examples include the following primer sets and the like. -Forward 5'-TCTTCACCCCTAACCAGCAGGAA-3' (SEQ ID NO: 12) -Reverse 5'-AGGACTGAAGTAAGTCCGATGC-3'(SEQ ID NO: 13)
  • the other steps are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a step of preparing a sample to be used in the detection step.
  • the method for evaluating female cancer can be suitably used as at least one of a method for diagnosing female cancer, a method for assisting diagnosis, and a method for predicting the onset of cancer.
  • the marker for evaluation of female cancer of the present invention contains at least a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1, and optionally contains other configurations.
  • the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is overexpressed in female cancer cells.
  • the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is involved in promoting the growth of female cancer cells and suppressing apoptosis through the regulation of expression of the RASSF5 gene and the ADORA1 gene. Therefore, a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 in a sample collected from an individual can be used as a marker for evaluation of female cancer.
  • the sample is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the female cancer is not particularly limited, and examples thereof include the same as those described in the item of ⁇ female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.
  • the other configurations are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose.
  • the marker for evaluating female cancer may include other markers for evaluating female cancer.
  • test examples of the present invention will be described below, but the present invention is not limited to these test examples.
  • RNA sequencing experiments using patient clinical information and clinical specimens were conducted with the approval of the Saitama Medical University International Medical Center IRB (# 13-165).
  • the RNA library was prepared with the SureSelect Strand Specific RNA library preparation kit (manufactured by Agilent), and RNA sequencing was performed using HiSeq2500® (manufactured by Illumina) under the condition of 100 bp Paired End.
  • the obtained FASTQ sequence file was aligned with the reference human genome (hg19) (S. Nagasawa, K. Ikeda, K. Horie-Inoue, S. Sato, A. Takeda, S. Takeda, K. Hasega. ... Systematic Identification of Characteristic Genes of Ovarian Clear Cell Carcinoma Compared with High-Grade Serous Carcinoma Based on RNA-Sequencing, Int J. Mol Sci, 20 (2019) 4330, doi: 10.3390 / ijms20184330)..
  • genes that positively or negatively correlate with OIN1 expression were determined by the correlation coefficient (r) and p-value.
  • genes having a positive and negative correlation were defined based on the value of the correlation coefficient (0.6 ⁇ r ⁇ 1 or -1 ⁇ r ⁇ ⁇ 0.45, respectively).
  • the biological pathways that significantly contain these genes are described in the database for analysis, visualization and integrated discovery (DAVID) Bioinformatics Resources 6.8 (https://daservers/svzysf.sc.s:/disks://david. analyzed.
  • ovarian cancer cells human ovarian cancer cells A2780, ES2, OV90, OVCAR3, RMG1 and SKOV3 were used.
  • endometrial cancer cell Ishikawa, which is a human endometrial cancer cell, was used.
  • BrC-PDC which is a human breast cancer cell established from a clinical sample of a breast cancer patient, was used.
  • OV90, OVCAR3, SKOV3, and Ishikawa were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 ⁇ g / mL streptomycin at 5% CO 2 , 37 ° C.
  • A2780 and RMG1 were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 ⁇ g / mL streptomycin at 5% CO 2 , 37 ° C.
  • ES2 was cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 ⁇ g / mL streptomycin under the conditions of 5% CO 2 and 37 ° C.
  • BrC-PDC is 8.8 ng / mL basic fibroblast growth factor (Thermo Fisher Scientific), 20 ⁇ mol / LY-27632 (Rho-associated coiled-coil forming kinase inhibitor), Thermo Fisher.
  • siRNA Small interference RNA
  • siControl used as a control was purchased from RNAi. The sequence of each siRNA is shown below.
  • RNA extraction and quantitative RT-PCR RNA was extracted from ovarian cancer cells or xenograft tumors derived from A2780 using ISOGEN reagent (manufactured by Nippon Gene Co., Ltd.). For crushing the tumor, Polytron PT3100 (manufactured by Kinematica) was used. Single-stranded cDNA was synthesized by reverse transcription from 1 ⁇ g of extracted RNA using SuperScript III (manufactured by Thermo Fisher Scientific) and a random hexamer primer.
  • ISOGEN reagent manufactured by Nippon Gene Co., Ltd.
  • Polytron PT3100 manufactured by Kinematica
  • Single-stranded cDNA was synthesized by reverse transcription from 1 ⁇ g of extracted RNA using SuperScript III (manufactured by Thermo Fisher Scientific) and a random hexamer primer.
  • qRT-PCR uses the prepared cDNA, the KAPA SYBR FAST qPCR kit (manufactured by KAPA Biosystems), and a set of gene-specific primers to be used in the StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific). I went there. RNA expression was analyzed by the ⁇ Ct method according to the StepOnePlus Real-Time PCR System product protocol, and the GAPDH gene expression was used for correction.
  • the primers used for qRT-PCR are as follows.
  • Cells were seeded in 96-well plate wells, 3,000 cells each for A2780 and RMG1, 1,000 cells each for SKOV3, and 2,000 cells each for Ishikawa. After culturing for 24 hours, cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific). The cells were collected 1 day, 3 days and 5 days after seeding. Transfection of a plasmid expressing the OIN1 gene or an empty vector was performed using FuGene HD Transfection Reagent (manufactured by Promega). The cells were collected 1 day and 3 days after seeding.
  • DNA assay To evaluate cell proliferation, the DNA of the cells in the wells was stained with Hoechst 33258 pentahydrate (Thermo Fisher Scientific, final concentration 5 ⁇ g / mL). The amount of DNA in each well was measured by a 2030 ARVO X5 Multilabel Plate Reader (PerkinElmer) (DNA assay).
  • BrC-PDC 4,000 cells are suspended in 240 ⁇ L of Opti-MEM medium (manufactured by Thermo Fisher Scientific) and seeded on a 24-well plate (Ultra-Low Attachment Multiple Well Plate, manufactured by Corning). board. Immediately thereafter, cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific). Six hours after transfection, 200 ⁇ L of StemPro hESC SFM medium (manufactured by Thermo Fisher Scientific) was added. The cells were collected 5 days after seeding, and the cells in each well were further divided into 5 wells of a 96-well plate and seeded.
  • Opti-MEM medium manufactured by Thermo Fisher Scientific
  • the amount of ATP of the cells in the well was measured using CellTiter - Glo 3D Cell Viability Assay (manufactured by Promega) and TriStar 2S LB 942 Multimode Reader (manufactured by Berthold Technologies). ..
  • mice Female nude mice (BALB / cAJcI-nu / nu) were purchased from Japan Claire Co., Ltd. A2780 (1 ⁇ 10 5 cells) and an equal amount of Matrix matrix (manufactured by Corning) were mixed and subcutaneously injected into the flank of a 10-week-old female nude mouse. Then, the mice were randomly divided into two groups.
  • SiControl or siOIN1 # 1 (5 ⁇ g each) was mixed with the transfection reagent GeneSilicer reagent (manufactured by Gene Therapy System) and injected into the tumor formed in mice twice a week.
  • the tumor volume was measured once a week and calculated by the formula of 0.5 ⁇ (diameter of the first axis) ⁇ (diameter of the second axis) ⁇ (diameter of the third axis).
  • Ovarian cancer cells are highly expressed in ovarian cancer intergenic noncoding RNA 1 (OIN1) >>
  • OIN1 ovarian cancer intergenic noncoding RNA 1
  • NONHSAT013448 in the NONCODE database (NONCODE. Archive online: http: //www.noncode.org/ (accessed on 31 May 2020)) was compared with the normal tissue of the ovary. It was revealed that it is highly expressed in clear cell ovarian cancer and highly atypical serous ovarian cancer (see FIGS. 1A and 1B).
  • FIG. 1A is a diagram showing the results of comparing the expression levels of long non-coding RNA (OIN1) registered as NONHSAT013448 in normal ovarian tissue, clear ovarian cell cancer, and highly atypical serous ovarian cancer. ..
  • the long non-coding RNA registered as NONHSAT013448 was higher in expression in clear ovarian cell carcinoma and highly atypical serous ovarian cancer compared to normal tissue.
  • the expression level of the long-chain non-coding RNA registered as NONHSAT013448 was calculated from the value of RPKM (reads per kilobase per million defined reads) (JY Wang, AQ Lu, LJ inLn, Inc.). ovarian cancer, Clin. Chim.
  • the long non-coding RNA registered as NONHSAT013448 is transcribed from the gene also registered as NONHSAG005930 in the NONCODE database.
  • the NONSAG005930 gene is present in the long arm of human chromosome 10 (10q21.1), and is a gene encoding a protein that is closest to upstream and downstream, protocadherin-related 15 (PCDH15) and mannose binding lectin 2 (MBL2). ) Are separated from each other by about 0.78 Mb and about 0.20 Mb, respectively. That is, since the NONHSAG005930 gene does not have a region overlapping with the gene encoding the protein, NONHSAT013448 is classified into the long intergenic noncoding RNA category among the long non-coding RNAs.
  • OIN1 ovarian cancer long intergenic noncoding RNA 1
  • FIG. 1B The upper figure of FIG. 1B is a diagram in which normal tissue of the ovary and RNA-seqing leads derived from specimens of clear cell ovarian cancer and highly atypical serous ovarian cancer are mapped to the locus of OIN1. Is a diagram showing that OIN1 is composed of two exons.
  • FIG. 1C is a diagram showing the results of analyzing the expression level of OIN1 in ovarian cancer cells by the qRT-PCR method and correcting for the expression level of GAPDH mRNA.
  • OIN1 was found to be highly expressed in A2780, SKOV3, and RMG1 and weakly expressed in OV90 cells (see FIG. 1C). Since OIN1 is highly expressed, A2780, SKOV3, and RMG1 were used for the functional analysis experiment of OIN1.
  • OIN1 promotes the growth of ovarian cancer cells and suppresses apoptosis
  • siRNA siOIN1 # 1 and siOIN1 # 2
  • FIG. 2A shows the knockdown efficiency of OIN1 by siRNA (siOIN1 # 1 and siOIN1 # 2) with respect to OIN1 in A2780 (left in FIG. 2A), SKOV3 (center in FIG. 2A), and qRT-PCR in RMG1 (right in FIG. 2A). It is a figure which shows the result of the analysis by.
  • *** represents p ⁇ 0.0001.
  • Statistical analysis was performed by two-way ANOVA.
  • ** represents p ⁇ 0.001
  • *** represents p ⁇ 0.0001.
  • Statistical analysis was performed by two-way ANOVA.
  • Figures 2C-2F show the results of analysis of apoptosis by a method that combines cell staining with PI and Annexin V and flow cytometry.
  • FIGS. 2C and 2E The results when A2780 is used as cells are shown in FIGS. 2C and 2E, and the results when SKOV3 is used as cells are shown in FIGS. 2D and 2F.
  • knockdown of OIN1 promotes apoptosis of A2780 and SKOV3 by cell staining and flow cytometry with PI and Annexin V.
  • FIGS. 2E and 2F the proportion of cells that have undergone apoptosis in A2780 and SKOV3 is quantified and shown in a graph.
  • ** represents p ⁇ 0.001
  • *** represents p ⁇ 0.0001.
  • Statistical analysis was performed by two-way ANOVA.
  • siOIN1 # 1 and siOIN1 # 2 increase the apoptosis of A2780 and SKOV3. Therefore, OIN1 was shown to promote the growth of ovarian cancer cells through suppression of apoptosis.
  • Table 1 shows a summary of genes whose expression pattern positively correlates with the expression pattern of OIN1 in specimens of highly atypical serous ovarian cancer and biological pathways rich in those genes. Shown below is a summary of genes whose expression pattern negatively correlates with the expression pattern of OIN1 in a sample of highly atypical serous ovarian cancer and a biological pathway rich in those genes.
  • the genes that positively correlate with the expression of OIN1 are biological such as “Inner cell mass cell proliferation”, “DNA replication initiation”, “Response to UV”, and “Response to X-ray”. It was allowed to accumulate in the pathway.
  • the genes that negatively correlate with the expression of OIN1 are "Intracellular signal transduction”, “Regulation of apoptosis”, “Cytoskeleton organization”, “Negative cell biology”, etc. Accumulated on the pathway.
  • the horizontal axis shows various genes, and the bar graphs in the genes show the case of transfecting siControl (left), the case of transfecting siOIN1 # 1 (center), and the case of transfecting siOIN1 # 2, respectively.
  • the result of (right) is shown, and * represents p ⁇ 0.05.
  • Statistical analysis was performed by two-way ANOVA.
  • the graph on the left shows the results for the RASSF5 gene
  • the graph on the right shows the results for the ADORA1 gene.
  • * represents p ⁇ 0.05
  • ** represents p ⁇ 0.01.
  • Statistical analysis was performed by two-way ANOVA.
  • RNA that the expression patterns of the genes RASSF5 and ADORA1 tend to be negatively correlated with the expression pattern of OIN1 in clinical specimens of highly atypical serous ovarian cancer. Shown from sequencing data.
  • RASSF5 and ADORA1 are downstream genes of OIN1.
  • RASSF5 binds to macrophage structuring 1/2 (MST1 / 2), causes activation of MST1 / 2, and affects cell proliferation and apoptosis (TJ). Liao, CJ Tsai, H. Jang, D. Fushman, R. Nussinov, RASSF5: An MST activator and tumor suppressor in vivo apoptosis. -224, doi: 10.1016 / j.sbi. 2016.09.001.).
  • RASSF5 has been reported to be involved in molecule-induced apoptosis such as tumor necrosis factor ⁇ (TNF- ⁇ ), TNF-related apoptosis-inducing led (TRAIL), and CD40 led (J. Park, S. I. Kang, S. Y. Lee, X. F. Zhang, MS Kim, LF Beers, DS Lim, J. Avruch, HS Kim, SB Lee, Tumor suppressor ras apoptosis domain family 5 (RASSF5 / NORE1) mediates death receptor lid-ind-indicated apoptosis, J. Biol. Chem. Elmetwalli, A.
  • TNF- ⁇ tumor necrosis factor ⁇
  • TRAIL TNF-related apoptosis-inducing led
  • CD40 led J. Park, S. I. Kang, S. Y. Lee, X. F. Zhang, MS Kim, LF Beers, DS Lim, J. Avruch, HS
  • ADORA1 is a 7-transmembrane G protein-coupled receptor and a receptor for adenosine in the extracellular space (MH Kazemi, S. Raofi Mohseni, M. Hojjat-Farsangi, E. Anvari, G. Ghamfarsa, H. Mohammadi, F. Jadidi-Niaargh, Adenosine and adenosine receptors in the immunopathogenesis in the imiunopathogenesis s. ). It has been reported that the function of ADORA1 in cancer differs depending on the type of cancer.
  • OIN1 suppresses apoptosis induced by RASSF5 and ADORA1 and promotes the growth of ovarian cancer.
  • OIN1 plays an important role in the growth of ovarian tumors in vivo
  • A2780 cells were mixed with Matrigel and injected subcutaneously into female nude mice.
  • siOIN1 # 1 was injected into the cancer twice a week and the growth of A2780-derived xenograft tumor was observed for 18 days.
  • FIGS. 4A-4F The results are shown in FIGS. 4A-4F.
  • FIG. 4A shows an example of an ovarian cancer xenograft tumor model mouse injected with siControl, and the lower figure shows an example of an ovarian cancer xenograft tumor model mouse injected with siOIN1 # 1.
  • FIG. 4B shows the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA.
  • Statistical analysis was performed by Student's t-test. The siRNA was injected into the formed xenograft tumor twice a week.
  • the graph in FIG. 4C shows the mean ⁇ SD of the tumor weight.
  • * represents p ⁇ 0.05.
  • Statistical analysis was performed by the Mann-Whitney U test.
  • siOIN1 # 1 was shown to significantly reduce the volume and weight of A2780-derived xenograft tumors.
  • FIGS. 4D-4F The results of examining the expression of OIN1, RASSF5, and ADORA1 in the tumor are shown in FIGS. 4D-4F.
  • FIG. 4D shows the results of analysis of the knockdown efficiency of OIN1 by injection of siOIN1 # 1 by qRT-PCR.
  • the relative expression level of OIN1 was corrected by the expression level of GAPDH mRNA and calculated, and the graph shows the average value ⁇ SD of the change in the expression level of OIN1 when compared with the siControl treatment conditions.
  • N 3 for siControl-treated tumors
  • n 3 for siOIN1 # 1 treated tumors
  • * represents p ⁇ 0.05.
  • Statistical analysis was performed by Student's t-test.
  • OIN1 regulates the expression of RASSF5 and ADORA1 to suppress apoptosis and promote the growth of ovarian cancer in vivo. Therefore, it was suggested that the double-stranded nucleic acid molecule of the present invention targeting OIN1 is useful as a nucleic acid drug discovery for female cancer.
  • OIN1 promotes the growth of endometrial cancer and breast cancer cells
  • siRNA siRNA
  • FIG. 5A shows the results of processing shiOIN1 # 1 or siOIN1 # 2 on Ishikawa and analyzing the knockdown efficiency of OIN1 by qRT-PCR.
  • ** represents p ⁇ 0.01.
  • Statistical analysis was performed by two-way ANOVA.
  • FIG. 5B shows the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of Ishikawa.
  • *** represents p ⁇ 0.0001.
  • Statistical analysis was performed by two-way ANOVA.
  • FIG. 5C shows the results of processing siOIN1 # 1 on BrC-PDC and analyzing the knockdown efficiency of OIN1 by qRT-PCR.
  • * represents p ⁇ 0.05.
  • Statistical analysis was performed by Student's t-test.
  • FIG. 5D shows the results of investigating that knockdown of OIN1 suppresses the proliferation of BrC-PDC by CellTiter-3D Cell Viability Assay (Promega).
  • ** represents p ⁇ 0.001.
  • Statistical analysis was performed by Student's t-test.
  • FIGS. 6C and 6F show the results of confirming changes in the expression of OIN1 RNA in A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector.
  • “Vector” shows an empty vector
  • "OIN1” shows the result when transfected with an OIN1 expression plasmid.
  • "***" in FIGS. 6C and 6F indicates p ⁇ 0.001 (Student's t-test).
  • FIGS. 6A and 6B “Vector” shows an empty vector, and "OIN1” shows the result when transfected with an OIN1 expression plasmid.
  • “*” in FIGS. 6A and 6B indicates p ⁇ 0.05, and "***” indicates p ⁇ 0.001 (Student's t-test).
  • FIGS. 6D A2780 cells, RASSF5)
  • FIG. 6G SKOV3 cells, RASSF5
  • FIG. 6H SKOV3 cells, ADORA1.
  • FIGS. 6D, 6E, 6G, and 6H "Vector” shows an empty vector
  • "OIN1” shows the result when transfected with an OIN1 expression plasmid.
  • “*" in FIGS. 6D, 6E, 6G, and 6H indicates p ⁇ 0.05
  • "**" indicates p ⁇ 0.01 (Student's t-test).
  • OIN1 is highly expressed in endometrial cancer
  • RPKM reads per kilobase per methylion complemented reads
  • OIN1 is important not only for ovarian cancer but also for the growth of female cancers such as breast cancer and endometrial cancer, which is an example of uterine cancer.
  • female cancers such as breast cancer and endometrial cancer, which is an example of uterine cancer.
  • the double-stranded nucleic acid molecule of the present invention can suppress the growth of not only ovarian cancer cells but also female cancer cells such as breast cancer cells and uterine body cancer cells, targeting OIN1. It was suggested that the double-stranded nucleic acid molecule could be applied to nucleic acid discovery of female cancer.
  • Examples of aspects of the present invention include the following. ⁇ 1> A double-stranded nucleic acid molecule for suppressing the expression of a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
  • B A double-stranded nucleic acid molecule comprising the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
  • ⁇ 2> The double-stranded nucleic acid molecule according to ⁇ 1>, which is either a double-stranded RNA or a double-stranded RNA-DNA chimera.
  • ⁇ 3> The double-stranded nucleic acid molecule according to any one of ⁇ 1> to ⁇ 2>, which is either siRNA or chimeric siRNA.
  • ⁇ 4> The double-stranded nucleic acid molecule according to any one of ⁇ 1> to ⁇ 3>, which is siRNA.
  • ⁇ 5> A DNA comprising a base sequence encoding the double-stranded nucleic acid molecule according to any one of ⁇ 1> to ⁇ 4>.
  • ⁇ 6> A vector comprising the DNA according to ⁇ 5>.
  • ⁇ 7> It is characterized by containing at least one of the double-stranded nucleic acid molecule according to any one of ⁇ 1> to ⁇ 4>, the DNA according to the above ⁇ 5>, and the vector according to the above ⁇ 6>. It is a female cancer cell growth inhibitor.
  • a method for suppressing the growth of female cancer cells which comprises allowing the female cancer cell to act on the female cancer cell growth inhibitor according to any one of ⁇ 7> to ⁇ 8>.
  • ⁇ 10> The method for suppressing the growth of female cancer cells according to ⁇ 9>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
  • the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
  • ⁇ 11> It is characterized by containing at least one of the double-stranded nucleic acid molecule according to any one of ⁇ 1> to ⁇ 4>, the DNA according to the above ⁇ 5>, and the vector according to the above ⁇ 6>. It is a tumor formation inhibitor for female cancer.
  • ⁇ 12> The tumor formation inhibitor for female cancer according to ⁇ 11>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
  • a method for suppressing tumor formation of female cancer which comprises allowing a tumor of female cancer to act on the tumor formation inhibitor of female cancer according to any one of ⁇ 11> to ⁇ 12>. .. ⁇ 14> The method for suppressing tumor formation of female cancer according to ⁇ 13>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
  • a drug for preventing or treating female cancer which is the female cancer cell growth inhibitor according to any one of ⁇ 7> to ⁇ 8> and any of the above ⁇ 11> to ⁇ 12>. It is a drug characterized by containing at least one of the tumor formation inhibitors for female cancers described in the above.
  • ⁇ 16> The drug according to ⁇ 15>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
  • a method for preventing or treating female cancer which comprises administering the drug according to any one of ⁇ 15> to ⁇ 16> to an individual.
  • ⁇ 19> Whether or not the subject suffers from female cancer using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index.
  • a method for evaluating female cancer which comprises evaluating whether or not the patient has a possibility of suffering from female cancer.
  • a marker for evaluation of female cancer which comprises a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.

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Abstract

Provided is a double-stranded nucleic acid molecule which is for inhibiting expression of long-chain noncoding RNA that comprises a base sequence represented by SEQ ID NO: 1 and which includes: (a) a sense strand that comprises a base sequence which corresponds to a target sequence comprising a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3; and (b) an antisense strand that comprises a base sequence which is complementary to the sense strand (a) and that forms, with the sense strand, a double strand.

Description

二本鎖核酸分子、ＤＮＡ、ベクター、女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤、医薬、及び長鎖非コードＲＮＡの利用Utilization of double-stranded nucleic acid molecules, DNA, vectors, female cancer cell growth inhibitors, tumor formation inhibitors for female cancers, pharmaceuticals, and long-chain non-coding RNAs

　本発明は、女性がんの予防乃至治療に好適に用いることができる二本鎖核酸分子、前記二本鎖核酸分子をコードする配列を含むＤＮＡ、前記ＤＮＡを含むベクター、前記二本鎖核酸分子、前記ＤＮＡ、及び前記ベクターの少なくともいずれかを含む女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤、前記女性がん細胞増殖抑制剤及び前記女性がんの腫瘍形成抑制剤の少なくともいずれかを含む医薬、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの有無又はその発現量を指標とする女性がんの評価方法、及び女性がんの評価用マーカーに関する。 The present invention is a double-stranded nucleic acid molecule that can be suitably used for the prevention or treatment of female cancer, a DNA containing a sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, and the double-stranded nucleic acid molecule. , At least one of the female cancer cell growth inhibitor or the female cancer tumor formation inhibitor, the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor, which contains at least one of the DNA and the vector. The present invention relates to a drug containing any of these, a method for evaluating female cancer using the presence or absence of a long-stranded non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 or the expression level thereof as an index, and a marker for evaluating female cancer.

　女性がんの１つである卵巣がんは、女性の罹患数の最も多いがんのひとつである（非特許文献１参照）。世界保健機関の外部機関である国際がん研究機関（ｔｈｅ　Ｉｎｔｅｒｎａｔｉｏｎａｌ　Ａｇｅｎｃｙ　ｆｏｒ　Ｒｅｓｅａｒｃｈ　ｏｎ　Ｃａｎｃｅｒ；　ＩＡＲＣ）の作成するデータベース（ＧＬＯＢＯＣＡＮ２０１８）によると、卵巣がんは世界における女性の罹患数が全がん種の中で８番目に多く、新規の罹患数および死亡数はそれぞれ２９５，４１４人と１８４，７７９人であると推定されている（非特許文献２参照）。卵巣がんの治療法は進歩し続けているが、女性がんにおいて卵巣がんの死亡率は依然として最も高い（非特許文献３参照）。早期に発見された卵巣がんについては治療が奏功する傾向にあるが、初期の段階の卵巣がんは症状に乏しく、症例の約６０％がより進行した段階で初めて診断される。これは卵巣がん患者の５年生存率が５０％を下回る一因となっている（非特許文献４～６参照）。そのため、卵巣がんの対策は喫緊の課題であり、卵巣がんの治療実績を改善するためには、新規のバイオマーカー・治療標的等が不可欠である。 Ovarian cancer, which is one of the female cancers, is one of the most prevalent cancers in women (see Non-Patent Document 1). According to the database (GLOBOCAN2018) created by the International Agency for Research on Cancer (IARC), which is an external organization of the World Health Organization, ovarian cancer affects all cancer types in women worldwide. It is the eighth highest among them, and the number of new cases and deaths is estimated to be 295,414 and 184,779, respectively (see Non-Patent Document 2). Although treatments for ovarian cancer continue to advance, ovarian cancer mortality remains the highest among female cancers (see Non-Patent Document 3). Treatment tends to be successful for early-detected ovarian cancer, but early-stage ovarian cancer is less symptomatic and is diagnosed only when about 60% of cases are more advanced. This contributes to the 5-year survival rate of patients with ovarian cancer falling below 50% (see Non-Patent Documents 4 to 6). Therefore, countermeasures for ovarian cancer are an urgent issue, and new biomarkers, therapeutic targets, etc. are indispensable for improving the treatment results of ovarian cancer.

　近年、ｃＤＮＡクローニングおよびＲＮＡシーケンシング技術の進歩に伴い、ヒトを含めた哺乳類の細胞では、ゲノムの約７０～９０％が転写され、タンパク質をコードしないさまざまな非コードＲＮＡ（ｎｏｎｃｏｄｉｎｇ　ＲＮＡ）が生じることが明らかにされた。さらに、非コードＲＮＡが、さまざま生命現象や、がんを含む疾患において重要な役割を果たすことも徐々に明らかとなってきている。 In recent years, with advances in cDNA cloning and RNA sequencing technology, about 70 to 90% of the genome is transcribed in mammalian cells, including humans, resulting in various non-coding RNAs that do not encode proteins. Was revealed. Furthermore, it is gradually becoming clear that non-coding RNA plays an important role in various life phenomena and diseases including cancer.

　非コードＲＮＡは長さによって分類され、特に２００塩基より長い非コードＲＮＡは長鎖非コードＲＮＡ（ｌｏｎｇ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡ；　ｌｎｃＲＮＡ）と定義される。ヒトのゲノムには膨大な数の長鎖非コードＲＮＡの遺伝子が存在するとされており、例えば、ヒトとマウスを含む１７種類の生物で発現する非コードＲＮＡのデータベースであるＮＯＮＣＯＤＥ（ｈｔｔｐ：／／ｗｗｗ．ｎｏｎｃｏｄｅ．ｏｒｇ／）は９６，３０８個にも及ぶヒトの長鎖非コードＲＮＡ遺伝子を登録している。長鎖非コードＲＮＡの大部分は依然として機能が明らかでないが、いくつかの長鎖非コードＲＮＡは、がんの病態生理において重要な役割を果たすことが示されている（非特許文献７～１０参照）。
　しかし、長鎖非コードＲＮＡの研究はまだ日が浅く、大部分の長鎖非コードＲＮＡの機能は未だ不明のままであり、卵巣がんなどの女性がんにおいてマーカーや治療標的として臨床応用されているものは存在しない。 Non-coding RNAs are classified by length, and in particular non-coding RNAs longer than 200 bases are defined as long noncoding RNAs (lncRNAs). It is said that a huge number of long-chain non-coding RNA genes exist in the human genome. For example, NONCODE (http://http: //), which is a database of non-coding RNA expressed in 17 kinds of organisms including human and mouse. www.noncode.org/) has registered as many as 96,308 human long non-coding RNA genes. Although most of the long non-coding RNAs are still unclear in function, some long non-coding RNAs have been shown to play an important role in the pathophysiology of cancer (Non-Patent Documents 7-10). reference).
However, research on long-chain non-coding RNAs is still young, and the function of most long-chain non-coding RNAs remains unknown, and is clinically applied as a marker or therapeutic target in female cancers such as ovarian cancer. There is nothing that is.

Ｚ．　Ｍｏｍｅｎｉｍｏｖａｈｅｄ，　Ａ．　Ｔｉｚｎｏｂａｉｋ，　Ｓ．　Ｔａｈｅｒｉ，　Ｈ．　Ｓａｌｅｈｉｎｉｙａ，　Ｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｉｎ　ｔｈｅ　ｗｏｒｌｄ：　ｅｐｉｄｅｍｉｏｌｏｇｙ　ａｎｄ　ｒｉｓｋ　ｆａｃｔｏｒｓ，　Ｉｎｔ．　Ｊ．　Ｗｏｍｅｎｓ　Ｈｅａｌｔｈ，　１１　（２０１９）　２８７－２９９，　ｄｏｉ：　１０．２１４７／ＩＪＷＨ．Ｓ１９７６０４．Z. Momenimoved, A. Tiznobak, S.A. Taheri, H. Salehiniya, Ovarian cancer in the world: epidemiology and risk factors, Int. J. Women's Health, 11 (2019) 287-299, doi: 10.2147 / IJWH. S197604. Ｆ．　Ｂｒａｙ，　Ｊ．　Ｆｅｒｌａｙ，　Ｉ．　Ｓｏｅｒｊｏｍａｔａｒａｍ，　Ｒ．Ｌ．　Ｓｉｅｇｅｌ，　Ｌ．Ａ．　Ｔｏｒｒｅ，　Ａ．　Ｊｅｍａｌ，　Ｇｌｏｂａｌ　ｃａｎｃｅｒ　ｓｔａｔｉｓｔｉｃｓ　２０１８：　ＧＬＯＢＯＣＡＮ　ｅｓｔｉｍａｔｅｓ　ｏｆ　ｉｎｃｉｄｅｎｃｅ　ａｎｄ　ｍｏｒｔａｌｉｔｙ　ｗｏｒｌｄｗｉｄｅ　ｆｏｒ　３６　ｃａｎｃｅｒｓ　ｉｎ　１８５　ｃｏｕｎｔｒｉｅｓ，　ＣＡ　Ｃａｎｃｅｒ　Ｊ．　Ｃｌｉｎ．，　６８　（２０１８）　３９４－４２４，　ｄｏｉ：　１０．３３２２／ｃａａｃ．２１４９２．F. Bray, J.M. Ferray, I. Soerjomataram, R.M. L. Siegel, L.M. A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldward for 36 cancers in 185 countries, CAC. Clin. , 68 (2018) 394-424, doi: 10.3322 / caac. 21492. Ｓ．Ｂ．　Ｃｏｂｕｒｎ，　Ｆ．　Ｂｒａｙ，　Ｍ．Ｅ．　Ｓｈｅｒｍａｎ，　Ｂ．　Ｔｒａｂｅｒｔ，　Ｉｎｔｅｒｎａｔｉｏｎａｌ　ｐａｔｔｅｒｎｓ　ａｎｄ　ｔｒｅｎｄｓ　ｉｎ　ｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｉｎｃｉｄｅｎｃｅ，　ｏｖｅｒａｌｌ　ａｎｄ　ｂｙ　ｈｉｓｔｏｌｏｇｉｃ　ｓｕｂｔｙｐｅ，　Ｉｎｔ．　Ｊ．　Ｃａｎｃｅｒ，　１４０　（２０１７）　２４５１－２４６０，　ｄｏｉ：　１０．１００２／ｉｊｃ．３０６７６．S. B. Coburn, F. Bray, M.M. E. Sherman, B. Travel, International patents and trends in ovarian cancer incidence, overall and by histological subtype, Int. J. Cancer, 140 (2017) 2451-2460, doi: 10.1002 / ijc. 30676. Ｃ．Ｖ．　Ｔｒｉｎｉｄａｄ，　Ａ．Ｌ．　Ｔｅｔｌｏｗ，　Ｌ．Ｅ．　Ｂａｎｔｉｓ，　Ａ．Ｋ．　Ｇｏｄｗｉｎ，　Ｒｅｄｕｃｉｎｇ　Ｏｖａｒｉａｎ　Ｃａｎｃｅｒ　Ｍｏｒｔａｌｉｔｙ　Ｔｈｒｏｕｇｈ　Ｅａｒｌｙ　Ｄｅｔｅｃｔｉｏｎ：　Ａｐｐｒｏａｃｈｅｓ　Ｕｓｉｎｇ　Ｃｉｒｃｕｌａｔｉｎｇ　Ｂｉｏｍａｒｋｅｒｓ，　Ｃａｎｃｅｒ　Ｐｒｅｖ．　Ｒｅｓ．　（Ｐｈｉｌａ），　１３　（２０２０）　２４１－２５２，　ｄｏｉ：　１０．１１５８／１９４０－６２０７．ＣＡＰＲ－１９－０１８４．C. V. Trinidad, A. L. Tetlow, L. E. Bantis, A. K. Goodwin, Reducing Ovarian Cancer Mortality Through Early Detection: Approaches Using Circulating Biomarkers, Cancer Prev. Res. (Phila), 13 (2020) 241-252, doi: 10.1158 / 1940-6207. CAPR-19-0184. Ｌ．Ａ．　Ｔｏｒｒｅ，　Ｂ．　Ｔｒａｂｅｒｔ，　Ｃ．Ｅ．　ＤｅＳａｎｔｉｓ，　Ｋ．Ｄ．　Ｍｉｌｌｅｒ，　Ｇ．　Ｓａｍｉｍｉ，　Ｃ．Ｄ．　Ｒｕｎｏｗｉｃｚ，　Ｍ．Ｍ．　Ｇａｕｄｅｔ，　Ａ．　Ｊｅｍａｌ，　Ｒ．Ｌ．　Ｓｉｅｇｅｌ，　Ｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｓｔａｔｉｓｔｉｃｓ，　２０１８，　ＣＡ　Ｃａｎｃｅｒ　Ｊ．　Ｃｌｉｎ．，　６８　（２０１８）　２８４－２９６，　ｄｏｉ：　１０．３３２２／ｃａａｃ．２１４５６．L. A. Torre, B. Travel, C.I. E. DeSantis, K.K. D. Miller, G.M. Samimi, C.I. D. Runowicz, M.D. M. Gaudet, A. Jemal, R. L. Siegel, Ovarian cancer statistics, 2018, CA Cancer J.C. Clin. , 68 (2018) 284-296, doi: 10.3322 / caac. 21456. Ｒ．Ｌ．　Ｓｉｅｇｅｌ，　Ｋ．Ｄ．　Ｍｉｌｌｅｒ，　Ａ．　Ｊｅｍａｌ，　Ｃａｎｃｅｒ　ｓｔａｔｉｓｔｉｃｓ，　２０１８，　ＣＡ　Ｃａｎｃｅｒ　Ｊ．　Ｃｌｉｎ．，　６８　（２０１８）　７－３０，　ｄｏｉ：　１０．３３２２／ｃａａｃ．２１４４２．R. L. Siegel, K.K. D. Miller, A. Jemal, Cancer statistics, 2018, CA Cancer J.C. Clin. , 68 (2018) 7-30, doi: 10.3322 / caac. 21442. Ａ．　Ｍｉｓａｗａ，　Ｋ．Ｉ．　Ｔａｋａｙａｍａ，　Ｓ．　Ｉｎｏｕｅ，　Ｌｏｎｇ　ｎｏｎ－ｃｏｄｉｎｇ　ＲＮＡｓ　ａｎｄ　ｐｒｏｓｔａｔｅ　ｃａｎｃｅｒ，　Ｃａｎｃｅｒ　Ｓｃｉ．，　１０８　（２０１７）　２１０７－２１１４，　ｄｏｉ：　１０．１１１１／ｃａｓ．１３３５２．A. Misawa, K.K. I. Takayama, S.A. Inoue, Long non-coding RNAs and prostate cancer, Cancer Sci. , 108 (2017) 2107-2114, doi: 10.1111 / cas. 13352. Ｙ．　Ｍｉｔｏｂｅ，　Ｋ．Ｉ．　Ｔａｋａｙａｍａ，　Ｋ．　Ｈｏｒｉｅ－Ｉｎｏｕｅ，　Ｓ．　Ｉｎｏｕｅ，　Ｐｒｏｓｔａｔｅ　ｃａｎｃｅｒ－ａｓｓｏｃｉａｔｅｄ　ｌｎｃＲＮＡｓ，　Ｃａｎｃｅｒ　Ｌｅｔｔ，　４１８　（２０１８）　１５９－１６６，　ｄｏｉ：　１０．１０１６／ｊ．ｃａｎｌｅｔ．２０１８．０１．０１２．Y. Mitobe, K.K. I. Takayama, K.K. Horie-Inoue, S.A. Inoue, Prostate cancer-assisted lncRNAs, Cancer Let, 418 (2018) 159-166, doi: 10.1016 / j. canlet. 2018.01.01.2. Ｇ．　Ａｒｕｎ，　Ｓ．Ｄ．　Ｄｉｅｒｍｅｉｅｒ，　Ｄ．Ｌ．　Ｓｐｅｃｔｏｒ，　Ｔｈｅｒａｐｅｕｔｉｃ　Ｔａｒｇｅｔｉｎｇ　ｏｆ　Ｌｏｎｇ　Ｎｏｎ－Ｃｏｄｉｎｇ　ＲＮＡｓ　ｉｎ　Ｃａｎｃｅｒ，　Ｔｒｅｎｄｓ　Ｍｏｌ．　Ｍｅｄ．，　２４　（２０１８）　２５７－２７７，　ｄｏｉ：　１０．１０１６／ｊ．ｍｏｌｍｅｄ．２０１８．０１．００１．G. Arun, S.A. D. Diermeier, D.I. L. Spector, Therapeutic Targeting of Long Non-coding RNAs in Cancer, Trends Mol. Med. , 24 (2018) 257-277, doi: 10.1016 / j. molmed. 2018.01.001. Ｔ．　Ｔａｋｅｉｗａ，　Ｋ．　Ｉｋｅｄａ，　Ｙ．　Ｍｉｔｏｂｅ，　Ｋ．　Ｈｏｒｉｅ－Ｉｎｏｕｅ，　Ｓ．　Ｉｎｏｕｅ，　Ｌｏｎｇ　Ｎｏｎｃｏｄｉｎｇ　ＲＮＡｓ　Ｉｎｖｏｌｖｅｄ　ｉｎ　ｔｈｅ　Ｅｎｄｏｃｒｉｎｅ　Ｔｈｅｒａｐｙ　Ｒｅｓｉｓｔａｎｃｅ　ｏｆ　Ｂｒｅａｓｔ　Ｃａｎｃｅｒ，　Ｃａｎｃｅｒｓ　（Ｂａｓｅｌ），　１２　（２０２０）　１４２４，　ｄｏｉ：　１０．３３９０／ｃａｎｃｅｒｓ１２０６１４２４．T. Takeiwa, K.K. Ikeda, Y. Mitobe, K.K. Horie-Inoue, S.A. Inoue, Long Noncoding RNAs Involved in the Endocline Therapy Response of Breast Cancer, Cancer, 12 (2020) 1424, cer1206: 1244, di14

　本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、女性がん細胞の増殖や女性がんの腫瘍形成を効果的に抑制することができ、女性がんの予防乃至治療に好適に用いることができる二本鎖核酸分子、前記二本鎖核酸分子をコードする塩基配列を含むＤＮＡ、前記ＤＮＡを含むベクター、前記二本鎖核酸分子、前記ＤＮＡ、及び前記ベクターの少なくともいずれかを含む女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤、前記女性がん細胞増殖抑制剤及び前記女性がんの腫瘍形成抑制剤の少なくともいずれかを含む医薬、女性がんの評価方法、及び女性がんの評価用マーカーを提供することを目的とする。 It is an object of the present invention to solve the above-mentioned conventional problems and to achieve the following objects. That is, the present invention is a double-stranded nucleic acid molecule that can effectively suppress the growth of female cancer cells and tumorigenesis of female cancer and can be suitably used for the prevention or treatment of female cancer. A female cancer cell growth inhibitor or a female cancer containing at least one of a DNA containing a base sequence encoding a double-stranded nucleic acid molecule, a vector containing the DNA, the double-stranded nucleic acid molecule, the DNA, and the vector. Provided are a drug containing at least one of the tumor formation inhibitor, the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor, a method for evaluating female cancer, and a marker for evaluating female cancer. The purpose is.

　本発明者らは、前記目的を達成するべく鋭意検討を行った結果、女性がんの検体において過剰発現する機能が未知の長鎖非コードＲＮＡ（配列番号：１、後述する「ｏｖａｒｉａｎ　ｌｏｎｇ　ｉｎｔｅｒｇｅｎｉｃ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡ　１」、以下、「ＯＩＮ１」と称することがある。）を同定した。また、前記長鎖非コードＲＮＡの発現を効率良く抑制する二本鎖核酸分子を設計し、前記二本鎖核酸分子をがん細胞や腫瘍に導入することにより、前記がん細胞の増殖及び前記腫瘍の形成が抑制されることを見出した。更に、前記二本鎖核酸分子を導入したがん細胞では、前記長鎖非コードＲＮＡの発現低下によりアポトーシスが誘導されることを見出した。 As a result of diligent studies to achieve the above object, the present inventors have conducted a long-chain non-coding RNA (SEQ ID NO: 1, SEQ ID NO: 1, which will be described later, "ovarian long intergenic noncoding") whose overexpressing function in female cancer specimens is unknown. RNA 1 ”, hereinafter sometimes referred to as“ OIN1 ”) was identified. Further, by designing a double-stranded nucleic acid molecule that efficiently suppresses the expression of the long-stranded non-coding RNA and introducing the double-stranded nucleic acid molecule into a cancer cell or a tumor, the growth of the cancer cell and the above-mentioned It was found that tumor formation was suppressed. Furthermore, it has been found that in cancer cells into which the double-stranded nucleic acid molecule has been introduced, apoptosis is induced by a decrease in the expression of the long-chain non-coding RNA.

　前記課題を解決するための手段としては、以下の通りである。即ち、
　＜１＞　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現を抑制するための二本鎖核酸分子であって、
（ａ）配列番号：２及び配列番号：３のいずれかで表される塩基配列からなる標的配列に対応する塩基配列を含むセンス鎖と、
（ｂ）前記（ａ）のセンス鎖と二本鎖を形成する該センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含むことを特徴とする二本鎖核酸分子である。
　＜２＞　前記＜１＞に記載の二本鎖核酸分子をコードする塩基配列を含むことを特徴とするＤＮＡである。
　＜３＞　前記＜２＞に記載のＤＮＡを含むことを特徴とするベクターである。
　＜４＞　前記＜１＞に記載の二本鎖核酸分子、前記＜２＞に記載のＤＮＡ、及び前記＜３＞に記載のベクターの少なくともいずれかを含むことを特徴とする女性がん細胞増殖抑制剤である。
　＜５＞　女性がん細胞に、前記＜４＞に記載の女性がん細胞増殖抑制剤を作用させることを特徴とする女性がん細胞の増殖抑制方法である。
　＜６＞　前記＜１＞に記載の二本鎖核酸分子、前記＜２＞に記載のＤＮＡ、及び前記＜３＞に記載のベクターの少なくともいずれかを含むことを特徴とする女性がんの腫瘍形成抑制剤である。
　＜７＞　女性がんの腫瘍に、前記＜６＞に記載の女性がんの腫瘍形成抑制剤を作用させることを特徴とする女性がんの腫瘍形成抑制方法である。
　＜８＞　女性がんを予防乃至治療するための医薬であって、前記＜４＞に記載の女性がん細胞増殖抑制剤及び前記＜６＞に記載の女性がんの腫瘍形成抑制剤の少なくともいずれかを含むことを特徴とする医薬である。
　＜９＞　個体に、前記＜８＞に記載の医薬を投与することを特徴とする女性がんの予防乃至治療方法である。
　＜１０＞　被検体由来の試料における配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの有無又はその発現量を指標として、前記被検体が女性がんに罹患しているか否か又は女性がんに罹患する可能性を有するか否かを評価することを含むことを特徴とする女性がんの評価方法である。
　＜１１＞　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを含むことを特徴とする女性がんの評価用マーカーである。 The means for solving the above problems are as follows. That is,
<1> A double-stranded nucleic acid molecule for suppressing the expression of a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
(A) A sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3.
(B) A double-stranded nucleic acid molecule comprising the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
<2> The DNA is characterized by containing a base sequence encoding the double-stranded nucleic acid molecule described in <1>.
<3> A vector comprising the DNA described in <2> above.
<4> Female cancer cell proliferation comprising at least one of the double-stranded nucleic acid molecule described in <1>, the DNA described in <2>, and the vector described in <3>. It is an inhibitor.
<5> A method for suppressing the growth of female cancer cells, which comprises allowing the female cancer cell growth inhibitor according to <4> to act on the female cancer cells.
<6> A tumor of female cancer characterized by containing at least one of the double-stranded nucleic acid molecule described in <1>, the DNA described in <2>, and the vector described in <3>. It is a formation inhibitor.
<7> A method for suppressing tumor formation of female cancer, which comprises allowing the tumor formation inhibitor for female cancer according to <6> to act on a tumor of female cancer.
<8> A drug for preventing or treating female cancer, which is at least one of the female cancer cell growth inhibitor according to <4> and the tumor formation inhibitor for female cancer according to <6>. It is a medicine characterized by containing any of them.
<9> A method for preventing or treating female cancer, which comprises administering the drug according to the above <8> to an individual.
<10> Whether or not the subject suffers from female cancer using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index. Alternatively, it is a method for evaluating female cancer, which comprises evaluating whether or not the patient has a possibility of suffering from female cancer.
<11> A marker for evaluation of female cancer, which comprises a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.

　本発明によると、従来における前記諸問題を解決することができ、女性がん細胞の増殖や女性がんの腫瘍形成を効果的に抑制することができ、女性がんの予防乃至治療に好適に用いることができる二本鎖核酸分子、前記二本鎖核酸分子をコードする塩基配列を含むＤＮＡ、前記ＤＮＡを含むベクター、前記二本鎖核酸分子、前記ＤＮＡ、及び前記ベクターの少なくともいずれかを含む女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤、前記女性がん細胞増殖抑制剤及び前記女性がんの腫瘍形成抑制剤の少なくともいずれかを含む医薬、女性がんの評価方法、及び女性がんの評価用マーカーを提供することができる。 According to the present invention, the above-mentioned problems in the prior art can be solved, the proliferation of female cancer cells and the tumor formation of female cancer can be effectively suppressed, and the present invention is suitable for the prevention or treatment of female cancer. A double-stranded nucleic acid molecule that can be used, a DNA containing a base sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, the double-stranded nucleic acid molecule, the DNA, and at least one of the vectors. A drug containing at least one of a female cancer cell growth inhibitor or a female cancer tumor formation inhibitor, the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor, a method for evaluating female cancer, And markers for evaluation of female cancer can be provided.

図１Ａは、卵巣の正常組織と、卵巣明細胞がん及び高異型度漿液性卵巣がんの検体におけるＯＩＮ１の発現量を比較した結果を示す図である。FIG. 1A is a diagram showing the results of comparing the expression level of OIN1 in the normal tissue of the ovary and the specimens of clear cell ovarian cancer and highly atypical serous ovarian cancer. 図１Ｂは、卵巣の正常組織と、卵巣明細胞がん及び高異型度漿液性卵巣がんの検体に由来するＲＮＡシーケンシングのリードをＯＩＮ１の遺伝子座へマッピングした結果を示す図である。FIG. 1B is a diagram showing the results of mapping RNA sequencing leads derived from normal ovarian tissue and specimens of clear ovarian cell cancer and highly atypical serous ovarian cancer to the OIN1 locus. 図１Ｃは、卵巣がん細胞におけるＯＩＮ１の発現をｑＲＴ－ＰＣＲ法により調べた結果を示す図である。FIG. 1C is a diagram showing the results of examining the expression of OIN1 in ovarian cancer cells by the qRT-PCR method. 図２Ａは、ＯＩＮ１に対するｓｉＲＮＡによるＯＩＮ１のノックダウン効率をＡ２７８０（左）、ＳＫＯＶ３（中央）、ＲＭＧ１（右）でｑＲＴ－ＰＣＲにより解析した結果を示す図である。FIG. 2A is a diagram showing the results of analysis of the knockdown efficiency of OIN1 by siRNA with respect to OIN1 by qRT-PCR with A2780 (left), SKOV3 (center), and RMG1 (right). 図２Ｂは、ＯＩＮ１のノックダウンがＡ２７８０（左）、ＳＫＯＶ３（中央）、ＲＭＧ１（右）の増殖を抑制することをＤＮＡアッセイにより調べた結果を示す図である。FIG. 2B is a diagram showing the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of A2780 (left), SKOV3 (center), and RMG1 (right). 図２Ｃは、Ａ２７８０細胞を用いてアポトーシスを解析した結果を示す図－１である。FIG. 2C is FIG. 1 showing the results of analysis of apoptosis using A2780 cells. 図２Ｄは、ＳＫＯＶ３細胞を用いてアポトーシスを解析した結果を示す図－１である。FIG. 2D is FIG. 1 showing the results of analysis of apoptosis using SKOV3 cells. 図２Ｅは、Ａ２７８０細胞を用いてアポトーシスを解析した結果を示す図－２である。FIG. 2E is FIG. 2 showing the results of analysis of apoptosis using A2780 cells. 図２Ｆは、ＳＫＯＶ３細胞を用いてアポトーシスを解析した結果を示す図－２である。FIG. 2F is FIG. 2 showing the results of analysis of apoptosis using SKOV3 cells. 図３Ａは、ＯＩＮ１のノックダウンが各種遺伝子の発現に与える影響に関して解析した結果を示す図－１である。FIG. 3A is a diagram showing the results of analysis regarding the effect of knockdown of OIN1 on the expression of various genes. 図３Ｂは、ＯＩＮ１のノックダウンが各種遺伝子の発現に与える影響に関して解析した結果を示す図－２である。FIG. 3B is FIG. 2 showing the results of analysis regarding the effect of knockdown of OIN1 on the expression of various genes. 図３Ｃは、高異型度漿液性卵巣がんの臨床検体において、ＲＡＳＳＦ５遺伝子（上図）とＡＤＯＲＡ１遺伝子（下図）の発現パターンをＲＮＡシーケンシングのデータから解析した結果を示す図である。FIG. 3C is a diagram showing the results of analysis of the expression patterns of the RASSF5 gene (upper figure) and the ADORA1 gene (lower figure) from RNA-Seqing data in a clinical specimen of highly atypical serous ovarian cancer. 図４Ａは、各ｓｉＲＮＡを注射した卵巣がん異種移植腫瘍モデルマウスの一例を示す図である。FIG. 4A is a diagram showing an example of an ovarian cancer xenograft tumor model mouse injected with each siRNA. 図４Ｂは、各ｓｉＲＮＡを注射したＡ２７８０由来異種移植腫瘍の増殖を調べた結果を示す図である。FIG. 4B is a diagram showing the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA. 図４Ｃは、異種移植腫瘍を切除し、重量を測定した結果を示す図である。FIG. 4C is a diagram showing the results of excising a xenograft tumor and measuring the weight. 図４Ｄは、各ｓｉＲＮＡを注射したＡ２７８０由来異種移植腫瘍におけるＯＩＮ１の発現を調べた結果を示す図である。FIG. 4D is a diagram showing the results of examining the expression of OIN1 in A2780-derived xenograft tumors injected with each siRNA. 図４Ｅは、各ｓｉＲＮＡを注射したＡ２７８０由来異種移植腫瘍におけるＲＡＳＳＦ５の発現を調べた結果を示す図である。FIG. 4E is a diagram showing the results of examining the expression of RASSF5 in A2780-derived xenograft tumors injected with each siRNA. 図４Ｆは、各ｓｉＲＮＡを注射したＡ２７８０由来異種移植腫瘍におけるＡＤＯＲＡ１の発現を調べた結果を示す図である。FIG. 4F is a diagram showing the results of examining the expression of ADORA1 in A2780-derived xenograft tumors injected with each siRNA. 図５Ａは、ＩｓｈｉｋａｗａにｓｉＯＩＮ１　＃１又はｓｉＯＩＮ１　＃２を処理し、ＯＩＮ１のノックダウン効率をｑＲＴ－ＰＣＲにより解析した結果を示す図である。FIG. 5A is a diagram showing the results of processing shiOIN1 # 1 or siOIN1 # 2 on Ishikawa and analyzing the knockdown efficiency of OIN1 by qRT-PCR. 図５Ｂは、ＯＩＮ１のノックダウンがＩｓｈｉｋａｗａの増殖を抑制することをＤＮＡアッセイにより調べた結果を示す図である。FIG. 5B is a diagram showing the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of Ishikawa. 図５Ｃは、ＢｒＣ－ＰＤＣにｓｉＯＩＮ１　＃１を処理し、ＯＩＮ１のノックダウン効率をｑＲＴ－ＰＣＲにより解析した結果を示す図である。FIG. 5C is a diagram showing the results of processing siOIN1 # 1 on BrC-PDC and analyzing the knockdown efficiency of OIN1 by qRT-PCR. 図５Ｄは、ＯＩＮ１のノックダウンがＢｒＣ－ＰＤＣの増殖を抑制することをＣｅｌｌＴｉｔｅｒ－３Ｄ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ　Ａｓｓａｙ（プロメガ社）により調べた結果を示す図である。FIG. 5D is a diagram showing the results of investigation by CellTiter-3D Cell Viability Assay (Promega) that knockdown of OIN1 suppresses the proliferation of BrC-PDC. 図６Ａは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０細胞の増殖を、ＤＮＡアッセイを用いて解析した結果を示す図である。FIG. 6A is a diagram showing the results of analysis of proliferation of A2780 cells transfected with the OIN1 expression plasmid or an empty vector using a DNA assay. 図６Ｂは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＳＫＯＶ３細胞の増殖を、ＤＮＡアッセイを用いて解析した結果を示す図である。FIG. 6B is a diagram showing the results of analysis of proliferation of SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector using a DNA assay. 図６Ｃは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０細胞におけるＯＩＮ１　ＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6C is a diagram showing the results of confirming changes in the expression of OIN1 RNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector. 図６Ｄは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０細胞におけるＲＡＳＳＦ５　ｍＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6D is a diagram showing the results of confirming changes in the expression of RASSF5 mRNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector. 図６Ｅは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０細胞におけるＡＤＯＲＡ１　ｍＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6E is a diagram showing the results of confirming changes in the expression of ADORA1 mRNA in A2780 cells transfected with the OIN1 expression plasmid or an empty vector. 図６Ｆは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＳＫＯＶ３細胞におけるＯＩＮ１　ＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6F is a diagram showing the results of confirming changes in the expression of OIN1 RNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector. 図６Ｇは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＳＫＯＶ３細胞におけるＲＡＳＳＦ５　ｍＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6G is a diagram showing the results of confirming changes in the expression of RASSF5 mRNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector. 図６Ｈは、ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＳＫＯＶ３細胞におけるＡＤＯＲＡ１　ｍＲＮＡの発現の変化を確認した結果を示す図である。FIG. 6H is a diagram showing the results of confirming changes in the expression of ADORA1 mRNA in SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector. 図７は、卵巣の正常組織と、子宮体がんの検体におけるＯＩＮ１の発現量を比較した結果を示す図である。FIG. 7 is a diagram showing the results of comparing the expression level of OIN1 in a normal tissue of an ovary and a sample of endometrial cancer.

（二本鎖核酸分子）
　本発明の二本鎖核酸分子は、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現を抑制するための二本鎖核酸分子であって、（ａ）配列番号：２及び配列番号：３のいずれかで表される塩基配列からなる標的配列に対応する塩基配列を含むセンス鎖と、（ｂ）前記（ａ）のセンス鎖と二本鎖を形成する該センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含む。
　なお、本発明において「二本鎖核酸分子」とは、センス鎖とアンチセンス鎖とがハイブリダイズしてなる二本鎖の核酸分子をいう。 (Double-stranded nucleic acid molecule)
The double-stranded nucleic acid molecule of the present invention is a double-stranded nucleic acid molecule for suppressing the expression of a long-chain non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1; (a) SEQ ID NO: 2 And the sense strand containing the base sequence corresponding to the target sequence consisting of the base sequence represented by any of SEQ ID NO: 3 and (b) the sense strand forming a double strand with the sense strand of (a) above. Includes an antisense strand containing a complementary base sequence.
In the present invention, the "double-stranded nucleic acid molecule" refers to a double-stranded nucleic acid molecule formed by hybridizing a sense strand and an antisense strand.

＜配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡ＞
　前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡは、ＮＯＮＣＯＤＥデータベース（ＮＯＮＣＯＤＥ．　Ａｖａｉｌａｂｌｅ　ｏｎｌｉｎｅ：　ｈｔｔｐ：／／ｗｗｗ．ｎｏｎｃｏｄｅ．ｏｒｇ／　（ａｃｃｅｓｓｅｄ　ｏｎ　３１　Ｍａｙ　２０２０）．）においてＮＯＮＨＳＡＴ０１３４４８として登録されていたが、これまで研究がなされておらず、その機能は不明であったものである。
　そこで、本発明者らは、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡをｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｌｏｎｇ　ｉｎｔｅｒｇｅｎｉｃ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡ　１（ＯＩＮ１）と命名した。
　前記ＯＩＮ１は、後述する試験例で示すように、女性がん細胞で過剰発現しており、女性がん細胞のアポトーシスを抑制し、女性がん細胞の増殖を亢進する機能を有する。 <Long-chain non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1>
The long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is referred to as NONHSAT103448 in the NONCODE database (NONCODE. Available on line: http: //www.noncode.org/ (accessed on 31 May 2020).). It was registered, but no research has been done so far, and its function is unknown.
Therefore, the present inventors have named a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 as ovarian cancer long intergenic noncoding RNA 1 (OIN1).
As shown in the test examples described later, OIN1 is overexpressed in female cancer cells and has a function of suppressing apoptosis of female cancer cells and promoting proliferation of female cancer cells.

　本発明では、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡが前記二本鎖核酸分子の標的となり、前記二本鎖核酸分子によってその発現が抑制されることから、本明細書中において前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを、前記二本鎖核酸分子の「標的ＲＮＡ」と称することがある。 In the present invention, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is targeted by the double-stranded nucleic acid molecule, and its expression is suppressed by the double-stranded nucleic acid molecule. In the specification, a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 may be referred to as a "target RNA" of the double-stranded nucleic acid molecule.

＜センス鎖、アンチセンス鎖＞
　本発明者らは、鋭意検討の結果、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの配列の中でも、ある特定の標的配列（配列番号：２及び配列番号：３のいずれかで表される塩基配列）に相補的な塩基配列を含むアンチセンス鎖を含む二本鎖核酸分子が、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡに対して顕著に優れた発現抑制効果を有することを見出した。したがって、本発明の二本鎖核酸分子は、（ａ）配列番号：２及び配列番号：３のいずれかで表される塩基配列からなる標的配列に対応する塩基配列を含むセンス鎖と、（ｂ）前記（ａ）のセンス鎖と二本鎖を形成する該センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含むものである。
　ここで、前記センス鎖及び前記アンチセンス鎖は、ＲＮＡ鎖であってもよいし、ＲＮＡ－ＤＮＡキメラ鎖であってもよい。前記センス鎖と前記アンチセンス鎖とは、互いにハイブリダイズすることで前記二本鎖核酸分子を形成することができる。 <Sense strand, antisense strand>
As a result of diligent studies, the present inventors have made a specific target sequence (SEQ ID NO: 2 and SEQ ID NO: 3) among the sequences of the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. A double-stranded nucleic acid molecule containing an antisense strand containing a base sequence complementary to the base sequence represented by any of the above is used for a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. It was found that it has a remarkably excellent expression inhibitory effect. Therefore, the double-stranded nucleic acid molecule of the present invention includes (a) a sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3 and (b). ) It includes the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
Here, the sense strand and the antisense strand may be an RNA strand or an RNA-DNA chimeric strand. The sense strand and the antisense strand can hybridize with each other to form the double-stranded nucleic acid molecule.

　なお、前記二本鎖核酸分子におけるセンス鎖は、前記標的配列に対応する塩基配列を含んでいればよく、その他の塩基配列を含んでいてもよく、前記標的配列に対応する塩基配列のみからなるものであってもよい。
　また、前記二本鎖核酸分子におけるアンチセンス鎖は、前記センス鎖とハイブリダイズすることができる程度に相補的な塩基配列を含んでいればよく、その他の塩基配列を含んでいてもよいが、前記センス鎖に相補的な塩基配列を７０％以上含むことが好ましく、８０％以上含むことがより好ましく、９０％以上含むことが更に好ましく、９５％以上含むことが特に好ましい。 The sense strand in the double-stranded nucleic acid molecule may contain a base sequence corresponding to the target sequence, may contain other base sequences, and consists only of the base sequence corresponding to the target sequence. It may be a thing.
Further, the antisense strand in the double-stranded nucleic acid molecule may contain a base sequence complementary to the extent that it can hybridize with the sense strand, and may contain other base sequences. The base sequence complementary to the sense strand is preferably contained in an amount of 70% or more, more preferably 80% or more, further preferably 90% or more, and particularly preferably 95% or more.

＜種類＞
　前記二本鎖核酸分子の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、二本鎖ＲＮＡ（ｄｏｕｂｌｅ－ｓｔｒａｎｄｅｄ　ＲＮＡ：ｄｓＲＮＡ）、二本鎖ＲＮＡ－ＤＮＡキメラなどが挙げられる。
　ここで、「二本鎖ＲＮＡ」とは、センス鎖及びアンチセンス鎖のいずれもがＲＮＡ配列で構成されてなる二本鎖核酸分子をいい、「二本鎖ＲＮＡ－ＤＮＡキメラ」とは、センス鎖及びアンチセンス鎖のいずれもがＲＮＡとＤＮＡとのキメラ配列で構成されてなる二本鎖核酸分子をいう。 <Type>
The type of the double-stranded nucleic acid molecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, double-stranded RNA (dsRNA), double-stranded RNA-DNA chimera, etc. Can be mentioned.
Here, the "double-stranded RNA" refers to a double-stranded nucleic acid molecule in which both the sense strand and the antisense strand are composed of RNA sequences, and the "double-stranded RNA-DNA chimera" refers to sense. A double-stranded nucleic acid molecule in which both the strand and the antisense strand are composed of a chimeric sequence of RNA and DNA.

　前記二本鎖ＲＮＡ及び二本鎖ＲＮＡ－ＤＮＡキメラは、ｓｉＲＮＡ（ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｉｎｇ　ＲＮＡ）若しくはキメラｓｉＲＮＡであることが好ましく、ｓｉＲＮＡであることがより好ましい。 The double-stranded RNA and double-stranded RNA-DNA chimera are preferably siRNA (small interfering RNA) or chimeric siRNA, and more preferably siRNA.

　ここで、ｓｉＲＮＡとは、１８塩基長～２９塩基長の小分子二本鎖ＲＮＡであり、前記ｓｉＲＮＡのアンチセンス鎖（ガイド鎖）と相補的な配列をもつ標的ＲＮＡを切断し、標的ＲＮＡの発現を抑制する機能を有する。
　前記ｓｉＲＮＡは、前記したようなセンス鎖及びアンチセンス鎖を有し、かつ前記標的ＲＮＡの発現を抑制し得るものであれば、その末端構造に特に制限はなく、目的に応じて適宜選択することができ、例えば、前記ｓｉＲＮＡは、平滑末端を有するものであってもよいし、突出末端（オーバーハング）を有するものであってもよい。中でも、前記ｓｉＲＮＡは、各鎖の３’末端が２塩基～６塩基突出した構造を有することが好ましく、各鎖の３’末端が２塩基突出した構造を有することがより好ましい。 Here, the siRNA is a small molecule double-stranded RNA having a length of 18 bases to 29 bases, and the target RNA having a sequence complementary to the antisense strand (guide strand) of the siRNA is cleaved to obtain the target RNA. It has a function of suppressing expression.
As long as the siRNA has the sense strand and the antisense strand as described above and can suppress the expression of the target RNA, the terminal structure thereof is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the siRNA may have a blunt end or a protruding end (overhang). Above all, the siRNA preferably has a structure in which the 3'end of each strand protrudes by 2 to 6 bases, and more preferably has a structure in which the 3'end of each strand protrudes by 2 bases.

　また、キメラｓｉＲＮＡとは、ｓｉＲＮＡのＲＮＡ配列の一部がＤＮＡに変換された、１８塩基長～２９塩基長の小分子二本鎖ＲＮＡ－ＤＮＡキメラをいう。中でも、ｓｉＲＮＡのセンス鎖の３’側の８塩基以内、及び、アンチセンス鎖の５’側の６塩基以内の塩基がＤＮＡに変換された、２１塩基長～２３塩基長の小分子二本鎖ＲＮＡ－ＤＮＡキメラであることが好ましい。前記キメラｓｉＲＮＡは、前記ｓｉＲＮＡと同様に、標的遺伝子の発現を抑制する機能を有する。なお、前記キメラｓｉＲＮＡには、ＤＮＡに変換された配列の一部をＲＮＡに再度変換した態様も含まれる。
　前記キメラｓｉＲＮＡの末端構造としても、前記ｓｉＲＮＡ同様、特に制限はなく、目的に応じて適宜選択することができ、例えば、平滑末端を有するものであってもよいし、突出末端（オーバーハング）を有するものであってもよい。 The chimeric siRNA refers to a small molecule double-stranded RNA-DNA chimera having a length of 18 bases to 29 bases in which a part of the RNA sequence of siRNA is converted into DNA. Among them, a small molecule double strand with a length of 21 to 23 bases, in which the bases within 8 bases on the 3'side of the sense strand of siRNA and within 6 bases on the 5'side of the antisense strand are converted into DNA. It is preferably an RNA-DNA chimera. The chimeric siRNA has a function of suppressing the expression of a target gene, similarly to the siRNA. The chimeric siRNA also includes an embodiment in which a part of the sequence converted into DNA is converted into RNA again.
The terminal structure of the chimeric siRNA is not particularly limited as in the case of the siRNA, and can be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang). It may have.

　前記ｓｉＲＮＡの具体例としては、下記のものなどが挙げられる。 Specific examples of the siRNA include the following.

　前記標的配列が、前記配列番号：２で表される塩基配列であるｓｉＲＮＡとしては、例えば、下記の配列番号：４のセンス鎖と配列番号：５のアンチセンス鎖とからなるｓｉＲＮＡが挙げられる。
　・　センス鎖
　　　５’－ＧＣＵＣＡＧＣＵＣＡＣＧＧＣＵＵＣＵＡＣＣ－３’（配列番号：４）
　・　アンチセンス鎖
　　　５’－ＵＡＧＡＡＧＣＣＧＵＧＡＧＣＵＧＡＧＣＵＣ－３’（配列番号：５） Examples of the siRNA whose target sequence is the base sequence represented by the SEQ ID NO: 2 include siRNA composed of the following sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 5.
-Sense strand 5'-GCUCAGCUCACGCGCUUCUC-3'(SEQ ID NO: 4)
-Antisense strand 5'-UAGAAGCCGUGAGCUGAGCUC-3'(SEQ ID NO: 5)

　また、前記標的配列が、前記配列番号：３で表される塩基配列であるｓｉＲＮＡとしては、例えば、下記の配列番号：６のセンス鎖と配列番号：７のアンチセンス鎖とからなるｓｉＲＮＡが挙げられる。
　・　センス鎖
　　　５’－ＧＡＣＡＧＧＡＧＡＣＵＣＣＡＧＡＡＡＡＧＧ－３’（配列番号：６）
　・　アンチセンス鎖
　　　５’－ＵＵＵＵＣＵＧＧＡＧＵＣＵＣＣＵＧＵＣＵＧ－３’（配列番号：７） Examples of the siRNA in which the target sequence is the base sequence represented by the SEQ ID NO: 3 include the siRNA composed of the following sense strand of SEQ ID NO: 6 and the antisense strand of SEQ ID NO: 7. Be done.
-Sense strand 5'-GACAGGAGACUCCAGAAAGG-3'(SEQ ID NO: 6)
-Antisense strand 5'-UUUUCUGGAGUCUCCUGUCUG-3'(SEQ ID NO: 7)

　また、前記二本鎖ＲＮＡは、ｓｈＲＮＡ（ｓｈｏｒｔ　ｈａｉｒｐｉｎ　ＲＮＡ）であってもよい。ここで、ｓｈＲＮＡとは、１８塩基～２９塩基程度のｄｓＲＮＡ領域と３塩基～９塩基程度のｌｏｏｐ領域を含む一本鎖ＲＮＡであるが、ｓｈＲＮＡは、生体内で発現されることにより、塩基対を形成してヘアピン状の二本鎖ＲＮＡとなる。その後、ｓｈＲＮＡはＤｉｃｅｒ（ＲＮａｓｅ　ＩＩＩ酵素）により切断されてｓｉＲＮＡとなり、標的ＲＮＡの発現抑制に機能することができる。
　前記ｓｈＲＮＡの末端構造としても、前記ｓｉＲＮＡ及び二本鎖ＲＮＡ－ＤＮＡキメラ同様、特に制限はなく、目的に応じて適宜選択することができ、例えば、平滑末端を有するものであってもよいし、突出末端（オーバーハング）を有するものであってもよい。 Further, the double-stranded RNA may be shRNA (short hairpin RNA). Here, the shRNA is a single-stranded RNA containing a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases, but the shRNA is a base pair when expressed in vivo. Is formed into a hairpin-shaped double-stranded RNA. After that, the shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to suppress the expression of the target RNA.
The terminal structure of the shRNA is not particularly limited as in the siRNA and the double-stranded RNA-DNA chimera, and can be appropriately selected depending on the intended purpose. For example, it may have a blunt end. It may have a protruding end (overhang).

＜修飾＞
　また、前記二本鎖核酸分子は、目的に応じて、適宜修飾を有していてもよい。例えば、核酸分解酵素（ヌクレアーゼ）に対する耐性を付与し、培養液中や生体中における安定性を向上させる等の目的から、前記二本鎖核酸分子に、２’Ｏ－ｍｅｔｈｙｌ化修飾や、ホスホロチオエート化（Ｓ化）修飾、ＬＮＡ（Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）修飾等を施すことができる。また、例えば、細胞への導入効率を高める等の目的から、前記二本鎖核酸分子のセンス鎖の５’端、或いは３’端に、ナノ粒子、コレステロール、細胞膜通過ペプチド等の修飾を施すこともできる。なお、前記二本鎖核酸分子にこのような修飾を施す方法としては、特に制限はなく、従来公知の手法を適宜利用することができる。 <Modification>
Further, the double-stranded nucleic acid molecule may have appropriate modifications depending on the purpose. For example, the double-stranded nucleic acid molecule is modified with 2'O-methyl or phosphorothioated for the purpose of imparting resistance to a nucleic acid-degrading enzyme (nuclease) and improving stability in a culture solution or in a living body. (S conversion) modification, LNA (Locked Nucleic Acid) modification and the like can be applied. Further, for example, for the purpose of increasing the efficiency of introduction into cells, the 5'end or 3'end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide or the like. You can also. The method for applying such a modification to the double-stranded nucleic acid molecule is not particularly limited, and a conventionally known method can be appropriately used.

＜入手方法＞
　前記二本鎖核酸分子の入手方法としては、特に制限はなく、それぞれ従来公知の手法に基づき作製することができる。
　例えば、前記ｓｉＲＮＡは、所望のセンス鎖とアンチセンス鎖とに相当する１８塩基長～２９塩基長の一本鎖ＲＮＡを、それぞれ既存のＤＮＡ／ＲＮＡ自動合成装置等を利用して化学的に合成し、それらをアニーリングすることにより作製することができる。また、アニーリング済の二本鎖ｓｉＲＮＡの市販品を入手することもできるし、ｓｉＲＮＡ合成受託会社に合成を依頼することにより入手することもできる。また、後述する本発明のベクターのような、所望のｓｉＲＮＡ発現ベクターを構築し、前記発現ベクターを細胞内に導入することにより、細胞内の反応を利用してｓｉＲＮＡを作製することもできる。
　また、前記キメラｓｉＲＮＡは、例えば、キメラ核酸分子であるセンス鎖とアンチセンス鎖とをそれぞれ化学合成し、それらをアニーリングすることにより、作製することができる。 <How to get>
The method for obtaining the double-stranded nucleic acid molecule is not particularly limited, and each can be produced based on a conventionally known method.
For example, the siRNA chemically synthesizes a single-stranded RNA having a length of 18 to 29 bases corresponding to a desired sense strand and an antisense strand, respectively, using an existing automatic DNA / RNA synthesizer or the like. And can be made by annealing them. In addition, a commercially available double-stranded siRNA that has been annealed can be obtained, or it can be obtained by requesting synthesis from a siRNA synthesis contractor. Further, by constructing a desired siRNA expression vector such as the vector of the present invention described later and introducing the expression vector into cells, siRNA can be produced by utilizing the intracellular reaction.
Further, the chimeric siRNA can be produced, for example, by chemically synthesizing a sense strand and an antisense strand, which are chimeric nucleic acid molecules, and annealing them.

（ＤＮＡ、ベクター）
　本発明のＤＮＡは、前記した本発明の二本鎖核酸分子をコードする塩基配列を含むＤＮＡであり、また、本発明のベクターは、前記ＤＮＡを含むベクターである。 (DNA, vector)
The DNA of the present invention is a DNA containing a base sequence encoding the double-stranded nucleic acid molecule of the present invention described above, and the vector of the present invention is a vector containing the DNA.

＜ＤＮＡ＞
　前記ＤＮＡとしては、前記した本発明の二本鎖核酸分子をコードする塩基配列を含むＤＮＡであれば特に制限はなく、目的に応じて適宜選択することができるが、前記二本鎖核酸分子をコードする塩基配列の上流（５’側）に、前記二本鎖核酸分子の転写を制御するためのプロモーター配列が連結されていることが好ましい。前記プロモーター配列としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ＣＭＶプロモーター等のｐｏｌ　ＩＩ系プロモーター、Ｈ１プロモーター、Ｕ６プロモーター等のｐｏｌ　ＩＩＩ系プロモーターなどが挙げられる。
　また、更に、前記二本鎖核酸分子をコードする塩基配列の下流（３’側）に、前記二本鎖核酸分子の転写を終結させるためのターミネーター配列が連結されていることがより好ましい。前記ターミネーター配列としても、特に制限はなく、目的に応じて適宜選択することができる。
　前記プロモーター配列、前記二本鎖核酸分子をコードする塩基配列、及び前記ターミネーター配列を含む転写ユニットは、前記ＤＮＡにおける好ましい一態様である。なお、前記転写ユニットは、従来公知の手法を用いて構築することができる。 <DNA>
The DNA is not particularly limited as long as it is a DNA containing a base sequence encoding the double-stranded nucleic acid molecule of the present invention, and can be appropriately selected depending on the intended purpose. It is preferable that a promoter sequence for controlling transcription of the double-stranded nucleic acid molecule is linked upstream (5'side) of the encoding base sequence. The promoter sequence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a pol II promoter such as a CMV promoter, a pol III promoter such as an H1 promoter and a U6 promoter, and the like.
Further, it is more preferable that the terminator sequence for terminating the transcription of the double-stranded nucleic acid molecule is linked downstream (3'side) of the base sequence encoding the double-stranded nucleic acid molecule. The terminator sequence is not particularly limited and may be appropriately selected depending on the intended purpose.
A transcription unit comprising the promoter sequence, the base sequence encoding the double-stranded nucleic acid molecule, and the terminator sequence is a preferred embodiment in the DNA. The transfer unit can be constructed by using a conventionally known method.

＜ベクター＞
　前記ベクターとしては、前記ＤＮＡを含むものであれば特に制限はなく、目的に応じて適宜選択することができ、例えば、プラスミドベクター、ウイルスベクターなどが挙げられる。前記ベクターは、前記二本鎖核酸分子を発現可能な発現ベクターであることが好ましい。
　前記二本鎖核酸分子の発現様式としては、特に制限はなく、目的に応じて適宜選択することができ、例えば二本鎖核酸分子としてｓｉＲＮＡを発現させる方法として、短い一本鎖ＲＮＡを二本発現させる方法（タンデム型）、ｓｈＲＮＡとしての一本鎖ＲＮＡを発現させる方法（ヘアピン型）等が挙げられる。
　前記タンデム型ｓｉＲＮＡ発現ベクターは、前記ｓｉＲＮＡを構成するセンス鎖をコードするＤＮＡ配列と、アンチセンス鎖をコードするＤＮＡ配列とを含み、かつ、各鎖をコードするＤＮＡ配列の上流（５’側）にプロモーター配列がそれぞれ連結され、また、各鎖をコードするＤＮＡ配列の下流（３’側）にターミネーター配列がそれぞれ連結されたＤＮＡを含む。
　また、前記ヘアピン型ｓｉＲＮＡ発現ベクターは、前記ｓｉＲＮＡを構成するセンス鎖をコードするＤＮＡ配列と、アンチセンス鎖をコードするＤＮＡ配列とが逆向きに配置され、前記センス鎖ＤＮＡ配列とアンチセンス鎖ＤＮＡ配列とがループ配列を介して接続されており、かつ、それらの上流（５’側）にプロモーター配列が、また、下流（３’側）にターミネーター配列が連結されたＤＮＡを含む。
　前記各ベクターは、従来公知の手法を用いて構築することができ、例えば、前記ＤＮＡを、予め制限酵素で切断したベクターの切断部位に連結（ライゲーション）することにより構築することができる。 <Vector>
The vector is not particularly limited as long as it contains the DNA, and can be appropriately selected depending on the intended purpose. Examples thereof include a plasmid vector and a virus vector. The vector is preferably an expression vector capable of expressing the double-stranded nucleic acid molecule.
The expression mode of the double-stranded nucleic acid molecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, as a method for expressing siRNA as a double-stranded nucleic acid molecule, two short single-stranded RNAs are used. Examples thereof include a method for expressing (tandem type), a method for expressing a single-stranded RNA as shRNA (hairpin type), and the like.
The tandem-type siRNA expression vector contains a DNA sequence encoding a sense strand constituting the siRNA and a DNA sequence encoding an antisense strand, and is upstream (5'side) of the DNA sequence encoding each strand. The promoter sequence is ligated to each, and the terminator sequence is ligated downstream (3'side) of the DNA sequence encoding each strand.
Further, in the hairpin-type siRNA expression vector, the DNA sequence encoding the sense strand constituting the siRNA and the DNA sequence encoding the antisense strand are arranged in opposite directions, and the sense strand DNA sequence and the antisense strand DNA are arranged in opposite directions. The sequences are connected via a loop sequence, and contain DNA to which the promoter sequence is linked upstream (5'side) and the terminator sequence is linked downstream (3'side).
Each of the vectors can be constructed by using a conventionally known method, for example, by ligating the DNA to a cleavage site of a vector previously cut with a restriction enzyme.

　前記ＤＮＡ又は前記ベクターを細胞に導入（トランスフェクト）することにより、プロモーターが活性化され、前記二本鎖核酸分子を生成することができる。例えば、前記タンデム型ベクターにおいては、前記ＤＮＡが細胞内で転写されることにより、センス鎖及びアンチセンス鎖が生成され、それらがハイブリダイズすることによりｓｉＲＮＡが生成される。前記ヘアピン型ベクターにおいては、前記ＤＮＡが細胞内で転写されることにより、まずヘアピン型ＲＮＡ（ｓｈＲＮＡ）が生成され、次いで、ダイサーによるプロセシングにより、ｓｉＲＮＡが生成される。 By introducing (transfecting) the DNA or the vector into a cell, the promoter can be activated and the double-stranded nucleic acid molecule can be produced. For example, in the tandem vector, the DNA is transcribed intracellularly to generate a sense strand and an antisense strand, and hybridizing them produces siRNA. In the hairpin-type vector, the DNA is transcribed intracellularly to first generate hairpin-type RNA (SHRNA), and then processing by a dicer produces siRNA.

（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）
　本発明の女性がん細胞増殖抑制剤は、女性がん細胞の増殖を抑制するためのものであり、前記した本発明の二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含み、更に必要に応じてその他の成分を含む。
　本発明の女性がんの腫瘍形成抑制剤は、女性がんの腫瘍の形成を抑制するためのものであり、前記した本発明の二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含み、更に必要に応じてその他の成分を含む。 (Female cancer cell growth inhibitor, female cancer tumor formation inhibitor)
The female cancer cell growth inhibitor of the present invention is for suppressing the growth of female cancer cells, and contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, and further. Includes other ingredients as needed.
The tumor formation inhibitor for female cancer of the present invention is for suppressing the formation of a tumor for female cancer, and contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above. , And other ingredients as needed.

＜二本鎖核酸分子、ＤＮＡ、ベクター＞
　前記二本鎖核酸分子の詳細としては、前記した本発明の二本鎖核酸分子の項目に記載した通りである。前記二本鎖核酸分子は、標的とする配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現を効果的に抑制することができるので、女性がん細胞の増殖を抑制するための前記女性がん細胞増殖抑制剤の有効成分として、又は女性がんの腫瘍の形成を抑制するための前記女性がんの腫瘍形成抑制剤の有効成分として好適である。また、前記ＤＮＡ、ベクターの詳細としても、前記した本発明のＤＮＡ、ベクターの項目に記載した通りである。
　前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤中の前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかの合計含有量としては、特に制限はなく、目的に応じて適宜選択することができる。また、前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤は、前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかそのものであってもよい。 <Double-stranded nucleic acid molecule, DNA, vector>
The details of the double-stranded nucleic acid molecule are as described in the above-mentioned item of the double-stranded nucleic acid molecule of the present invention. Since the double-stranded nucleic acid molecule can effectively suppress the expression of long-chain non-coding RNA consisting of the base sequence represented by the target SEQ ID NO: 1, it suppresses the proliferation of female cancer cells. It is suitable as an active ingredient of the female cancer cell proliferation inhibitor for the purpose of, or as an active ingredient of the tumor formation inhibitor of the female cancer for suppressing the formation of a female cancer tumor. Further, the details of the DNA and the vector are as described in the above-mentioned items of the DNA and the vector of the present invention.
The total content of at least one of the double-stranded nucleic acid molecule, DNA, and vector in the female cancer cell growth inhibitor or the tumor formation inhibitor of female cancer is not particularly limited, and may vary depending on the intended purpose. It can be selected as appropriate. Further, the female cancer cell growth inhibitor or the female cancer tumor formation inhibitor may be at least one of the double-stranded nucleic acid molecule, DNA, and vector itself.

＜その他の成分＞
　前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを所望の濃度に希釈するための生理食塩水、培養液等の希釈用剤や、対象とする細胞や腫瘍内に前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを導入（トランスフェクト）するためのトランスフェクション試薬などが挙げられる。
　前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤中の前記その他の成分の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。 <Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, physiology for diluting at least one of the double-stranded nucleic acid molecule, DNA, and vector to a desired concentration. Examples thereof include diluting agents such as saline solution and culture solution, and transfection reagents for introducing (transfecting) at least one of the double-stranded nucleic acid molecule, DNA, and vector into a target cell or tumor. Be done.
The content of the other components in the female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer is not particularly limited and may be appropriately selected depending on the intended purpose.

＜女性がん＞
　前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤の適用対象となる女性がんとしては、特に制限はなく、目的に応じて適宜選択することができるが、卵巣がん、子宮がん（子宮体がん、子宮頸がんを含む）、及び乳がんの少なくともいずれかが好適に挙げられる。
　前記卵巣がん、子宮がん、及び乳がんの種類としては、特に制限はなく、目的に応じて適宜選択することができる。 <Women's cancer>
The female cancer to which the female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer is applied is not particularly limited and may be appropriately selected depending on the intended purpose, but ovarian cancer and uterus. At least one of cancer (including uterine body cancer and cervical cancer) and breast cancer is preferably mentioned.
The types of ovarian cancer, uterine cancer, and breast cancer are not particularly limited and may be appropriately selected depending on the intended purpose.

　前記女性がん細胞は、体外で培養されている細胞であってもよいし、個体の体内に存在する細胞であってもよい。 The female cancer cell may be a cell cultured outside the body or a cell existing in the body of an individual.

＜作用＞
　前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤は、例えば、女性がん細胞又は女性がんの腫瘍に導入することによって、前記女性がん細胞又は前記女性がんの腫瘍に作用させることができる。前記導入の方法としては、特に制限はなく、従来公知の手法の中から目的に応じて適宜選択することができ、例えば、トランスフェクション試薬を用いる方法、エレクトロポレーションによる方法、磁気粒子を用いる方法、ウイルス感染を利用する方法、注射により注入する方法などが挙げられる。
　前記女性がん細胞又は前記女性がんの腫瘍に対して作用させる前記女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤の量としては、特に制限はなく、細胞や腫瘍の種類や所望の効果の程度等に応じて適宜選択することができるが、例えば、１×１０６個の細胞数に対し、有効成分（前記二本鎖核酸分子）の量として、０．１μｇ程度が好ましく、５μｇ程度がより好ましく、１５μｇ程度が特に好ましい。 <Action>
The female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor can be introduced into, for example, a female cancer cell or a female cancer tumor to cause the female cancer cell or the female cancer tumor. Can act. The method of introduction is not particularly limited and may be appropriately selected from conventionally known methods according to the purpose. For example, a method using a transfection reagent, a method using electroporation, or a method using magnetic particles. , A method using virus infection, a method of injecting by injection, etc.
The amount of the female cancer cell proliferation inhibitor or the tumor formation inhibitor of female cancer acting on the female cancer cell or the tumor of the female cancer is not particularly limited, and the type of cell or tumor may be used. It can be appropriately selected depending on the degree of desired effect, etc., but for example, the amount of the active ingredient (the double-stranded nucleic acid molecule) is preferably about 0.1 μg with respect to the number of cells of 1 × 10 6 . About 5 μg is more preferable, and about 15 μg is particularly preferable.

　本発明の女性がん細胞増殖抑制剤又は女性がんの腫瘍形成抑制剤は、それ自体を単独で使用してもよいし、両者を併用してもよいし、他の女性がん治療薬と組み合わせて使用してもよい。 The female cancer cell growth inhibitor or the tumor formation inhibitor for female cancer of the present invention may be used alone, in combination with each other, or with other therapeutic agents for female cancer. It may be used in combination.

（女性がん細胞の増殖抑制方法、女性がんの腫瘍形成抑制方法）
　前記女性がん細胞増殖抑制剤は、前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含むので、女性がん細胞に作用させることにより、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現抑制を介して、女性がん細胞の増殖を効果的に抑制することができる。したがって、本発明は、女性がん細胞に、前記女性がん細胞増殖抑制剤を作用させることを特徴とする女性がん細胞の増殖抑制方法にも関する。
　また、前記女性がんの腫瘍形成抑制剤は、前記二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含むので、女性がんの腫瘍に作用させることにより、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現抑制を介して、女性がんの腫瘍の形成を効果的に抑制することができる。したがって、本発明は、女性がんの腫瘍に、前記女性がんの腫瘍形成抑制剤を作用させることを特徴とする女性がんの腫瘍形成抑制方法にも関する。
　前記女性がんとしては、特に制限はなく、上記した（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の＜女性がん＞の項目に記載したものと同様のものが挙げられる。
　また、前記女性がん細胞の増殖抑制方法又は女性がんの腫瘍形成抑制方法では、他の女性がん治療薬を更に作用させてもよい。 (Method of suppressing the growth of female cancer cells, method of suppressing tumor formation of female cancer)
Since the female cancer cell growth inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, the base sequence represented by the above-mentioned SEQ ID NO: 1 is expressed by acting on female cancer cells. It is possible to effectively suppress the growth of female cancer cells through the suppression of the expression of long-chain non-coding RNA consisting of. Therefore, the present invention also relates to a method for suppressing the growth of female cancer cells, which comprises allowing the female cancer cell growth inhibitor to act on female cancer cells.
Further, since the tumor formation inhibitor for female cancer contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, it is represented by the above-mentioned SEQ ID NO: 1 by acting on the tumor of female cancer. The formation of tumors of female cancer can be effectively suppressed through the suppression of the expression of long-chain non-coding RNA consisting of the base sequence. Therefore, the present invention also relates to a method for suppressing tumor formation in female cancer, which comprises allowing the tumor formation inhibitor for female cancer to act on a tumor in female cancer.
The female cancer is not particularly limited, and examples thereof include the same as those described in the item of <female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.
Further, in the method for suppressing the growth of female cancer cells or the method for suppressing tumor formation of female cancer, another female cancer therapeutic agent may be further acted upon.

（医薬）
　本発明の医薬は、女性がんを予防乃至治療するための医薬であって、前記した本発明の女性がん細胞増殖抑制剤及び女性がんの腫瘍形成抑制剤の少なくともいずれかを含み、更に必要に応じてその他の成分を含む。
　本発明において、前記予防とは、女性がんの発症や再発を防止することをいい、前記治療とは、がんの症状を治したり、和らげたり、進行を防いだりすることをいう。
　前記女性がんとしては、特に制限はなく、上記した（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の＜女性がん＞の項目に記載したものと同様のものが挙げられる。 (Pharmaceutical)
The medicine of the present invention is a medicine for preventing or treating female cancer, and includes at least one of the above-mentioned female cancer cell growth inhibitor and female cancer tumor formation inhibitor of the present invention, and further. Contains other ingredients as needed.
In the present invention, the prevention means to prevent the onset and recurrence of female cancer, and the treatment means to cure, relieve, or prevent the progression of cancer symptoms.
The female cancer is not particularly limited, and examples thereof include the same as those described in the item of <female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.

＜女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤＞
　前記女性がん細胞増殖抑制剤及び女性がんの腫瘍形成抑制剤の詳細としては、前記した本発明の（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の項目に記載した通りである。 <Female cancer cell growth inhibitor, female cancer tumor formation inhibitor>
Details of the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor are described in the above-mentioned item of the present invention (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). It's a street.

　前記女性がん細胞増殖抑制剤は、前記した本発明の二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含むので、標的とする前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現抑制を介して、女性がん細胞の増殖を効果的に抑制することができる。
　即ち、前記女性がん細胞増殖抑制剤は、女性がんを予防乃至治療するための医薬として好適に利用可能である。 Since the female cancer cell growth inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, the length consisting of the base sequence represented by the above-mentioned SEQ ID NO: 1 as a target. The growth of female cancer cells can be effectively suppressed through the suppression of the expression of non-coding RNA.
That is, the female cancer cell growth inhibitor can be suitably used as a medicine for preventing or treating female cancer.

　前記女性がんの腫瘍形成抑制剤は、前記した本発明の二本鎖核酸分子、ＤＮＡ、及びベクターの少なくともいずれかを含むので、標的とする前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現抑制を介して、女性がんの腫瘍の形成を効果的に抑制することができる。
　即ち、前記女性がんの腫瘍形成抑制剤は、女性がんを予防乃至治療するための医薬として好適に利用可能である。 Since the tumor formation inhibitor for female cancer contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, it comprises the base sequence represented by the above-mentioned SEQ ID NO: 1 as a target. Through suppression of the expression of long-chain non-coding RNA, tumor formation of female cancer can be effectively suppressed.
That is, the tumor formation inhibitor for female cancer can be suitably used as a medicine for preventing or treating female cancer.

　前記医薬中の前記女性がん細胞増殖抑制剤及び女性がんの腫瘍形成抑制剤の少なくともいずれかの合計含有量としては、特に制限はなく、目的に応じて適宜選択することができる。また、前記医薬は、前記女性がん細胞増殖抑制剤及び女性がんの腫瘍形成抑制剤の少なくともいずれかのみからなるものであってもよい。 The total content of at least one of the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor in the drug is not particularly limited and may be appropriately selected depending on the intended purpose. Further, the drug may consist of at least one of the female cancer cell growth inhibitor and the female cancer tumor formation inhibitor.

　ここで、前記医薬の有効成分となる前記二本鎖核酸分子としては、非修飾の状態の二本鎖核酸分子そのものを用いてもよいが、適切に予防乃至治療効果が得られるよう、生体への投与に適した形態の二本鎖核酸分子を用いることが好ましい。
　例えば、前記二本鎖核酸分子は、生体内における二本鎖核酸分子の安定性を高めることができる点で、修飾が施されていることが好ましい。前記二本鎖核酸分子に施し得る修飾の種類としては、特に制限はなく、例えば、２’Ｏ－ｍｅｔｈｙｌ化修飾、ホスホロチオエート化（Ｓ化）修飾、ＬＮＡ（Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）修飾などが挙げられる。また、標的細胞への導入効率を高める等の目的から、例えば、前記二本鎖核酸分子のセンス鎖の５’端、或いは３’端に、ナノ粒子、コレステロール、細胞膜通過ペプチド等の修飾を施すこともまた好ましい。前記二本鎖核酸分子に前記修飾を施す方法としては、特に制限はなく、従来公知の手法を適宜利用することができる。
　また、前記二本鎖核酸分子は、標的細胞への導入効率を高めることができる点で、リポソームや高分子マトリックス等と複合体を形成していることも好ましい。前記複合体を形成する方法としても、特に制限はなく、従来公知の手法を適宜利用することができる。 Here, as the double-stranded nucleic acid molecule that is the active ingredient of the pharmaceutical, the double-stranded nucleic acid molecule itself in an unmodified state may be used, but it is applied to a living body so that an appropriate preventive or therapeutic effect can be obtained. It is preferable to use a double-stranded nucleic acid molecule in a form suitable for administration of.
For example, the double-stranded nucleic acid molecule is preferably modified in that the stability of the double-stranded nucleic acid molecule in a living body can be enhanced. The type of modification that can be applied to the double-stranded nucleic acid molecule is not particularly limited, and examples thereof include 2'O-methyl modification, phosphorothioate (S) modification, and LNA (Locked Nucleic Acid) modification. Further, for the purpose of increasing the efficiency of introduction into the target cell, for example, the 5'end or 3'end of the sense strand of the double-stranded nucleic acid molecule is modified with nanoparticles, cholesterol, a cell membrane-passing peptide or the like. It is also preferable. The method for applying the modification to the double-stranded nucleic acid molecule is not particularly limited, and a conventionally known method can be appropriately used.
Further, it is also preferable that the double-stranded nucleic acid molecule forms a complex with a liposome, a polymer matrix, or the like in that the introduction efficiency into the target cell can be enhanced. The method for forming the complex is not particularly limited, and a conventionally known method can be appropriately used.

＜その他の成分＞
　前記その他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、医薬的に許容され得る担体などが挙げられる。前記担体としても、特に制限はなく、例えば、剤型等に応じて適宜選択することができる。また、前記医薬中の前記その他の成分の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。 <Other ingredients>
The other components are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a pharmaceutically acceptable carrier. The carrier is also not particularly limited and may be appropriately selected depending on, for example, a dosage form or the like. Further, the content of the other component in the medicine is not particularly limited and may be appropriately selected depending on the intended purpose.

＜剤型＞
　前記医薬の剤型としては、特に制限はなく、例えば、所望の投与方法に応じて適宜選択することができ、例えば、経口固形剤（錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等）、経口液剤（内服液剤、シロップ剤、エリキシル剤等）、注射剤（溶液、懸濁液、用事溶解用固形剤等）、軟膏剤、貼付剤、ゲル剤、クリーム剤、外用散剤、スプレー剤、吸入散剤などが挙げられる。 <Dosage form>
The dosage form of the pharmaceutical is not particularly limited and may be appropriately selected depending on the desired administration method, for example, an oral solid preparation (tablet, coated tablet, granule, powder, capsule, etc.). Oral solutions (oral solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solids for errands, etc.), ointments, patches, gels, creams, external powders, sprays, inhalations. Examples include powders.

　前記経口固形剤としては、例えば、前記有効成分に、賦形剤、更には必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等の添加剤を加え、常法により製造することができる。
　前記賦形剤としては、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸などが挙げられる。前記結合剤としては、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどが挙げられる。前記崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖などが挙げられる。前記滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコールなどが挙げられる。前記着色剤としては、例えば、酸化チタン、酸化鉄などが挙げられる。前記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。 As the oral solid preparation, for example, an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / flavoring agent, etc. are added to the active ingredient, and a conventional method is used. Can be manufactured by
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Be done. Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, lactose and the like. Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

　前記経口液剤としては、例えば、前記有効成分に、矯味・矯臭剤、緩衝剤、安定化剤等の添加剤を加え、常法により製造することができる。
　前記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。前記緩衝剤としては、例えば、クエン酸ナトリウムなどが挙げられる。前記安定化剤としては、例えば、トラガント、アラビアゴム、ゼラチンなどが挙げられる。 As the oral solution, for example, it can be produced by a conventional method by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to the active ingredient.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include tragant, gum arabic, gelatin and the like.

　前記注射剤としては、例えば、前記有効成分に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下用、筋肉内用、静脈内用等の注射剤を製造することができる。
　前記ｐＨ調節剤及び前記緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。前記安定化剤としては、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸などが挙げられる。前記等張化剤としては、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。前記局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカインなどが挙げられる。 As the injection, for example, a pH regulator, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the active ingredient, and the injection is subcutaneously, intramuscularly, or intravenously used by a conventional method. Etc. can be produced.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.

　前記軟膏剤としては、例えば、前記有効成分に、公知の基剤、安定剤、湿潤剤、保存剤等を配合し、常法により混合し、製造することができる。
　前記基剤としては、例えば、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィンなどが挙げられる。前記保存剤としては、例えば、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピルなどが挙げられる。 As the ointment, for example, a known base, stabilizer, wetting agent, preservative and the like can be blended with the active ingredient and mixed by a conventional method to produce the ointment.
Examples of the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.

　前記貼付剤としては、例えば、公知の支持体に前記軟膏剤としてのクリーム剤、ゲル剤、ペースト剤等を、常法により塗布し、製造することができる。
　前記支持体としては、例えば、綿、スフ、化学繊維からなる織布、不織布、軟質塩化ビニル、ポリエチレン、ポリウレタン等のフィルム、発泡体シートなどが挙げられる。 As the patch, for example, a cream, a gel, a paste, or the like as the ointment can be applied to a known support by a conventional method to produce the patch.
Examples of the support include cotton, rayon, woven fabric made of chemical fibers, non-woven fabric, soft vinyl chloride, polyethylene, polyurethane and other films, foam sheets and the like.

＜投与＞
　前記医薬は、女性がんの予防乃至治療に好適である。したがって、前記医薬は、女性がんに罹患した又は罹患した可能性を有する個体に投与することにより好適に使用することができる。 <Administration>
The drug is suitable for the prevention or treatment of female cancer. Therefore, the said medicine can be suitably used by administering it to an individual who has or may have suffered from female cancer.

　前記医薬の投与対象個体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、マウス、ラット、ウシ、ブタ、サル、イヌ、ネコなどが挙げられるが、これらの中でも、ヒトが特に好ましい。 The individual to be administered with the drug is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs and cats. Of these, humans are particularly preferred.

　前記医薬の投与方法としては、特に制限はなく、例えば、前記医薬の剤型、疾患の種類、患者の状態等に応じて、局所投与、全身投与のいずれかを選択することができる。例えば、局所投与においては、前記医薬の有効成分（前記二本鎖核酸分子）を、所望の部位（例えば、腫瘍部位）に直接注入することにより投与することができる。前記注入には、注射等の従来公知の手法を適宜利用することができる。また、全身投与（例えば、経口投与、腹腔内投与、血液中への投与等）においては、前記医薬の有効成分（前記二本鎖核酸分子）が所望の部位（例えば、腫瘍部位）まで安定に、かつ効率良く送達されるよう、従来公知の薬剤送達技術を適宜応用することが好ましい。 The method of administering the drug is not particularly limited, and for example, either local administration or systemic administration can be selected according to the dosage form of the drug, the type of disease, the condition of the patient, and the like. For example, in local administration, the active ingredient of the drug (the double-stranded nucleic acid molecule) can be administered by directly injecting it into a desired site (for example, a tumor site). For the injection, a conventionally known method such as injection can be appropriately used. Further, in systemic administration (for example, oral administration, intraperitoneal administration, administration into blood, etc.), the active ingredient of the drug (the double-stranded nucleic acid molecule) is stably delivered to a desired site (for example, a tumor site). It is preferable to appropriately apply a conventionally known drug delivery technique so that the drug can be delivered efficiently.

　前記医薬の投与量としては、特に制限はなく、投与対象である患者の年齢、体重、所望の効果の程度等に応じて適宜選択することができるが、例えば、成人への１日の投与あたり、有効成分（前記二本鎖核酸分子）の量として、１ｍｇ～１００ｍｇが好ましい。
　また、前記医薬の投与回数としても、特に制限はなく、投与対象である患者の年齢、体重、所望の効果の程度等に応じて、適宜選択することができる。 The dose of the drug is not particularly limited and may be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the patient to be administered. For example, per daily administration to an adult. The amount of the active ingredient (the double-stranded nucleic acid molecule) is preferably 1 mg to 100 mg.
The number of administrations of the drug is not particularly limited, and can be appropriately selected depending on the age, body weight, degree of desired effect, and the like of the patient to be administered.

　前記医薬の投与時期としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、疾患に対して、予防的に投与されてもよいし、治療的に投与されてもよい。中でも、前記医薬は、女性がん細胞の増殖を阻害する効果や、女性がんの腫瘍の形成を抑制する効果に優れることから、前記医薬は前記疾患の出来る限り早期の段階に投与されることが望ましいと考えられる。 The administration time of the drug is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be administered prophylactically or therapeutically for a disease. Above all, since the drug is excellent in the effect of inhibiting the growth of female cancer cells and the effect of suppressing the formation of tumors of female cancer, the drug should be administered at the earliest possible stage of the disease. Is considered desirable.

　本発明の医薬は、他の女性がん治療薬と組み合わせて使用してもよい。 The pharmaceutical product of the present invention may be used in combination with other female cancer therapeutic agents.

（女性がんの予防乃至治療方法）
　前記医薬は、前記女性がん細胞増殖抑制剤及び女性がんの腫瘍形成抑制剤の少なくともいずれかを含むので、女性がんに罹患した又は罹患した可能性を有する個体に投与することにより、標的とする配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現抑制を介して、女性がん細胞の増殖及び女性がんの腫瘍の形成の少なくともいずれかを効果的に抑制し、女性がんを予防乃至治療することができる。したがって、本発明は、個体に、前記医薬を投与することを特徴とする女性がんの予防乃至治療方法にも関する。
　前記女性がんとしては、特に制限はなく、上記した（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の＜女性がん＞の項目に記載したものと同様のものが挙げられる。
　また、前記予防乃至治療方法では、他の女性がん治療薬を更に投与してもよい。 (Prevention or treatment method for female cancer)
Since the drug comprises at least one of the female cancer cell proliferation inhibitor and the female cancer tumor growth inhibitor, it is targeted by administration to an individual who has or may have suffered from female cancer. It effectively suppresses at least one of the proliferation of female cancer cells and the formation of female cancer tumors through the suppression of the expression of long-chain non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. , Can prevent or treat female cancer. Therefore, the present invention also relates to a method for preventing or treating female cancer, which comprises administering the above-mentioned medicine to an individual.
The female cancer is not particularly limited, and examples thereof include the same as those described in the item of <female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.
Further, in the preventive or therapeutic method, another female cancer therapeutic agent may be further administered.

（女性がんの評価方法）
　本発明の女性がんの評価方法は、評価工程を少なくとも含み、必要に応じて検出工程などのその他の工程を含む。
　前記女性がんとしては、特に制限はなく、上記した（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の＜女性がん＞の項目に記載したものと同様のものが挙げられる。 (Evaluation method for female cancer)
The method for evaluating female cancer of the present invention includes at least an evaluation step, and if necessary, includes other steps such as a detection step.
The female cancer is not particularly limited, and examples thereof include the same as those described in the item of <female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.

＜評価工程＞
　前記評価工程は、被検体由来の試料における配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの有無又はその発現量を指標として、前記被検体が女性がんに罹患しているか否か又は女性がんに罹患する可能性を有するか否かを評価する工程である。 <Evaluation process>
In the evaluation step, whether or not the subject suffers from female cancer using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index. It is a step of evaluating whether or not a woman has a possibility of developing cancer.

－被検体由来の試料－
　前記被検体由来の試料としては、対象とする個体から調製したものであれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、病変部位の細胞や組織、血液などが挙げられる。前記試料は、更にＲＮＡ調製処理などが施されてもよい。前記試料は、１種のみを使用してもよいし、２種以上を使用してもよい。 -Sample derived from the subject-
The sample derived from the subject is not particularly limited as long as it is prepared from a target individual, and can be appropriately selected according to the purpose. Examples thereof include cells, tissues, and blood at the lesion site. Be done. The sample may be further subjected to RNA preparation treatment or the like. As the sample, only one kind may be used, or two or more kinds may be used.

　前記被検体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、マウス、ラット、ウシ、ブタ、サル、イヌ、ネコなどが挙げられるが、これらの中でも、ヒトが特に好ましい。 The subject is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs and cats, and among these, humans. Is particularly preferable.

－配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡ－
　前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡは、上記した本発明の（二本鎖核酸分子）の＜配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡ＞の項目に記載したとおりである。 -Long-chain non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1-
The long-chain non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is the long-chain non-coding RNA consisting of the base sequence represented by <SEQ ID NO: 1 of the above-mentioned (double-stranded nucleic acid molecule) of the present invention. As described in the item of RNA>.

－評価－
　前記試料中に前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡが検出された場合、前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現量が健常個体由来の試料中における発現量と比べて多い場合、又は前記配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現量が女性がんに罹患している個体由来の試料における発現量と比べて同等以上の場合には、前記個体が、女性がんに罹患している又は罹患する可能性を有すると評価する。 -evaluation-
When a long-chain non-coding RNA consisting of the base sequence represented by the SEQ ID NO: 1 is detected in the sample, the expression level of the long-chain non-coding RNA consisting of the base sequence represented by the SEQ ID NO: 1 is increased. A sample derived from an individual suffering from female cancer when the expression level is higher than the expression level in a sample derived from a healthy individual, or when the expression level of a long non-coding RNA consisting of the base sequence represented by the above-mentioned SEQ ID NO: 1 is high. When the expression level is equal to or higher than the expression level in the above individual, it is evaluated that the individual has or may have female cancer.

＜検出工程＞
　前記検出工程は、個体から調製した試料中における配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを検出する工程である。 <Detection process>
The detection step is a step of detecting a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 in a sample prepared from an individual.

－検出－
　前記検出の方法としては、特に制限はなく、公知の方法を目的に応じて適宜選択することができ、例えば、ｑＲＴ－ＰＣＲによる方法、ＲＮＡシーケンスによる方法などが挙げられる。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。 -detection-
The detection method is not particularly limited, and a known method can be appropriately selected depending on the intended purpose. Examples thereof include a method by qRT-PCR and a method by RNA sequencing. These may be used alone or in combination of two or more.

　前記ＰＣＲに用いるプライマーセットとしては、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの塩基配列を特異的に増幅できるものであれば、特に制限はなく、目的に応じて適宜選択することができる。 The primer set used for the PCR is not particularly limited as long as it can specifically amplify the base sequence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. You can choose.

　前記プライマーセットの一例としては、下記のプライマーセットなどが挙げられる。
　・　フォワード
　　　５’－ＴＣＴＴＣＡＣＣＣＣＴＡＡＣＣＡＧＣＡＧＧＡＡ－３’（配列番号：１２）
　・　リバース
　　　５’－ＡＧＧＡＣＴＧＡＡＧＴＡＡＧＴＣＣＴＧＡＴＧＣ－３’（配列番号：１３） Examples of the primer set include the following primer sets and the like.
-Forward 5'-TCTTCACCCCTAACCAGCAGGAA-3' (SEQ ID NO: 12)
-Reverse 5'-AGGACTGAAGTAAGTCCGATGC-3'(SEQ ID NO: 13)

＜その他の工程＞
　前記その他の工程としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記検出工程に用いる試料を調製する工程などが挙げられる。 <Other processes>
The other steps are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a step of preparing a sample to be used in the detection step.

　前記女性がんの評価方法は、女性がんの診断方法、診断補助方法、及び発症予測方法の少なくともいずれかとして、好適に用いることができる。 The method for evaluating female cancer can be suitably used as at least one of a method for diagnosing female cancer, a method for assisting diagnosis, and a method for predicting the onset of cancer.

（女性がんの評価用マーカー）
　本発明の女性がんの評価用マーカーは、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを少なくとも含み、必要に応じてその他の構成を含む。 (Marker for evaluation of female cancer)
The marker for evaluation of female cancer of the present invention contains at least a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1, and optionally contains other configurations.

　後述する試験例で示すように、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡは、女性がん細胞において過剰発現している。また、配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡは、ＲＡＳＳＦ５遺伝子やＡＤＯＲＡ１遺伝子の発現制御を介して、女性がん細胞の増殖促進とアポトーシス抑制に関与している。
　したがって、個体から採取された試料などにおける配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡは、女性がんの評価用マーカーとして用いることができる。
　前記試料としては、特に制限はなく、目的に応じて適宜選択することができる。 As shown in the test examples described later, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is overexpressed in female cancer cells. In addition, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is involved in promoting the growth of female cancer cells and suppressing apoptosis through the regulation of expression of the RASSF5 gene and the ADORA1 gene.
Therefore, a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1 in a sample collected from an individual can be used as a marker for evaluation of female cancer.
The sample is not particularly limited and may be appropriately selected depending on the intended purpose.

　前記女性がんとしては、特に制限はなく、上記した（女性がん細胞増殖抑制剤、女性がんの腫瘍形成抑制剤）の＜女性がん＞の項目に記載したものと同様のものが挙げられる。 The female cancer is not particularly limited, and examples thereof include the same as those described in the item of <female cancer> in the above-mentioned (female cancer cell growth inhibitor, female cancer tumor formation inhibitor). Be done.

　前記その他の構成としては、本発明の効果を損なわない限り、特に制限はなく、目的に応じて適宜選択することができる。前記女性がんの評価用マーカーは、その他の女性がんの評価用マーカーを含んでいてもよい。 The other configurations are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected depending on the intended purpose. The marker for evaluating female cancer may include other markers for evaluating female cancer.

　以下に本発明の試験例を説明するが、本発明は、これらの試験例に何ら限定されるものではない。 The test examples of the present invention will be described below, but the present invention is not limited to these test examples.

（試験例１）
＜材料と方法＞
＜＜正常卵巣組織、卵巣腫瘍、及び子宮体がんの臨床検体を用いたＲＮＡシーケンシング解析＞＞
　患者の臨床情報及び臨床検体を使用したＲＮＡシーケンシング実験については、埼玉医科大学国際医療センターＩＲＢの承認のもと行われた（＃１３－１６５）。高異型度漿液性卵巣がん（ｈｉｇｈ－ｇｒａｄｅ　ｓｅｒｏｕｓ　ｏｖａｒｉａｎ　ｃａｒｃｉｎｏｍａ；　ＨＧＳＯＣ）の標本（ｎ＝１５）、卵巣明細胞がん（ｏｖａｒｉａｎ　ｃｌｅａｒ　ｃｅｌｌ　ｃａｒｃｉｎｏｍａ；　ＯＣＣＣ）の標本（ｎ＝６）、正常組織（Ｎｏｒｍａｌ）の標本（ｎ＝６）、又は子宮体がんの標本（ｎ＝３４）は、インフォームドコンセントを得て原発性卵巣腫瘍の手術を受けた患者から入手した（＃１２－０９６）。 (Test Example 1)
<Materials and methods>
<< RNA-Seqing Analysis Using Clinical Specimens of Normal Ovarian Tissue, Ovarian Tumor, and Endometrial Cancer >>
RNA sequencing experiments using patient clinical information and clinical specimens were conducted with the approval of the Saitama Medical University International Medical Center IRB (# 13-165). High-grade serous ovarian cancer (HGSOC) specimen (n = 15), ovarian clear cell cancer (OCCC) specimen (n = 6), normal tissue (n = 6) Normal) specimens (n = 6) or uterine body cancer specimens (n = 34) were obtained from patients who had informed outlets and underwent surgery for primary ovarian tumors (# 12-096).

　ＲＮＡは、ＮｕｃｌｅｏＳｐｉｎ　ＲＮＡ（タカラ社製）を使用して、凍結直後の組織から抽出した。ＲＮＡのクオリティーはバイオアナライザー（Ａｇｉｌｅｎｔ社製）で解析し、全てのＲＮＡのＲＩＮ値は７以上であった。
　ＲＮＡライブラリをＳｕｒｅＳｅｌｅｃｔ　Ｓｔｒａｎｄ　Ｓｐｅｃｉｆｉｃ　ＲＮＡ　ライブラリ　調製キット（Ａｇｉｌｅｎｔ社製）で調製し、ＨｉＳｅｑ２５００（登録商標）（Ｉｌｌｕｍｉｎａ社製）を用いて１００ｂｐ　Ｐａｉｒｅｄ　Ｅｎｄの条件でＲＮＡシーケンスを施行した。 RNA was extracted from tissue immediately after freezing using NucleoSpin RNA (manufactured by Takara). The quality of RNA was analyzed by a bioanalyzer (manufactured by Agilent), and the RIN value of all RNA was 7 or more.
The RNA library was prepared with the SureSelect Strand Specific RNA library preparation kit (manufactured by Agilent), and RNA sequencing was performed using HiSeq2500® (manufactured by Illumina) under the condition of 100 bp Paired End.

　得られたＦＡＳＴＱシークエンスファイルをリファレンスヒトゲノム（ｈｇ１９）にアラインした（Ｓ．　Ｎａｇａｓａｗａ，　Ｋ．　Ｉｋｅｄａ，　Ｋ．　Ｈｏｒｉｅ－Ｉｎｏｕｅ，　Ｓ．　Ｓａｔｏ，　Ａ．　Ｉｔａｋｕｒａ，　Ｓ．　Ｔａｋｅｄａ，　Ｋ．　Ｈａｓｅｇａｗａ，　Ｓ．　Ｉｎｏｕｅ，　Ｓｙｓｔｅｍａｔｉｃ　Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ　ｏｆ　Ｃｈａｒａｃｔｅｒｉｓｔｉｃ　Ｇｅｎｅｓ　ｏｆ　Ｏｖａｒｉａｎ　Ｃｌｅａｒ　Ｃｅｌｌ　Ｃａｒｃｉｎｏｍａ　Ｃｏｍｐａｒｅｄ　ｗｉｔｈ　Ｈｉｇｈ－Ｇｒａｄｅ　Ｓｅｒｏｕｓ　Ｃａｒｃｉｎｏｍａ　Ｂａｓｅｄ　ｏｎ　ＲＮＡ－Ｓｅｑｕｅｎｃｉｎｇ，　Ｉｎｔ．　Ｊ．　Ｍｏｌ．　Ｓｃｉ．，　２０　（２０１９）　４３３０，　ｄｏｉ：　１０．３３９０／ｉｊｍｓ２０１８４３３０．）。 The obtained FASTQ sequence file was aligned with the reference human genome (hg19) (S. Nagasawa, K. Ikeda, K. Horie-Inoue, S. Sato, A. Takeda, S. Takeda, K. Hasega. ... Systematic Identification of Characteristic Genes of Ovarian Clear Cell Carcinoma Compared with High-Grade Serous Carcinoma Based on RNA-Sequencing, Int J. Mol Sci, 20 (2019) 4330, doi: 10.3390 / ijms20184330)..

　高異型度漿液性卵巣がんの検体において、ＯＩＮ１の発現と正又は負に相関する遺伝子を相関係数（ｒ）とｐ値により求めた。
　次に、相関係数の値に基づいて、正と負の相関を持つ遺伝子を定義した（それぞれ０．６≦ｒ≦１、又は－１≦ｒ≦－０．４５）。これらの遺伝子が有意に含まれる生物学的パスウェイをｔｈｅ　ｄａｔａｂａｓｅ　ｆｏｒ　ａｎｎｏｔａｔｉｏｎ，　ｖｉｓｕａｌｉｚａｔｉｏｎ　ａｎｄ　ｉｎｔｅｇｒａｔｅｄ　ｄｉｓｃｏｖｅｒｙ（ＤＡＶＩＤ）　Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ　Ｒｅｓｏｕｒｃｅｓ　６．８（ｈｔｔｐｓ：／／ｄａｖｉｄ．ｎｃｉｆｃｒｆ．ｇｏｖ／ｓｕｍｍａｒｙ．ｊｓｐ）のデータベースを用いて分析した。 In a sample of highly atypical serous ovarian cancer, genes that positively or negatively correlate with OIN1 expression were determined by the correlation coefficient (r) and p-value.
Next, genes having a positive and negative correlation were defined based on the value of the correlation coefficient (0.6 ≦ r ≦ 1 or -1 ≦ r ≦ −0.45, respectively). The biological pathways that significantly contain these genes are described in the database for analysis, visualization and integrated discovery (DAVID) Bioinformatics Resources 6.8 (https://daservers/svzysf.sc.s:/disks://david. analyzed.

＜＜細胞培養＞＞
　卵巣がん細胞として、ヒト卵巣がん細胞であるＡ２７８０、ＥＳ２、ＯＶ９０、ＯＶＣＡＲ３、ＲＭＧ１、ＳＫＯＶ３を使用した。
　子宮体がん細胞として、ヒト子宮体がん細胞であるＩｓｈｉｋａｗａを使用した。
　乳がん細胞として、乳がん患者の臨床検体より樹立されたヒト乳がん細胞であるＢｒＣ－ＰＤＣを使用した。 << Cell Culture >>
As ovarian cancer cells, human ovarian cancer cells A2780, ES2, OV90, OVCAR3, RMG1 and SKOV3 were used.
As the endometrial cancer cell, Ishikawa, which is a human endometrial cancer cell, was used.
As a breast cancer cell, BrC-PDC, which is a human breast cancer cell established from a clinical sample of a breast cancer patient, was used.

　ＯＶ９０、ＯＶＣＡＲ３、ＳＫＯＶ３、及びＩｓｈｉｋａｗａは、１０％ウシ胎児血清、１００Ｕ／ｍＬペニシリン、及び１００μｇ／ｍＬストレプトマイシンを含むＤＭＥＭ培地で、５％ＣＯ２、３７℃の条件で培養した。
　Ａ２７８０及びＲＭＧ１は、１０％ウシ胎児血清、１００Ｕ／ｍＬペニシリン、及び１００μｇ／ｍＬストレプトマイシンを含むＲＰＭＩ　１６４０培地で、５％ＣＯ２、３７℃の条件で培養した。
　ＥＳ２は、１０％ウシ胎児血清、１００Ｕ／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシンを含むＤＭＥＭ／Ｆ１２培地で、５％ＣＯ２、３７℃の条件で培養した。
　ＢｒＣ－ＰＤＣは、８．８ｎｇ／ｍＬ　ｂａｓｉｃ　ｆｉｂｒｏｂｌａｓｔ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ（サーモフィッシャーサイエンティフィック社製）、２０μｍｏｌ／Ｌ　Ｙ－２７６３２（Ｒｈｏ－ａｓｓｏｃｉａｔｅｄ　ｃｏｉｌｅｄ－ｃｏｉｌ　ｆｏｒｍｉｎｇ　ｋｉｎａｓｅ阻害剤、サーモフィッシャーサイエンティフィック社製）、５０Ｕ／ｍＬペニシリン、５０μｇ／ｍＬストレプトマイシンを含むＳｔｅｍＰｒｏ　ｈＥＳＣ　ＳＦＭ培地（サーモフィッシャーサイエンティフィック社製）を用い、Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ　Ｍｕｌｔｉｐｌｅ　Ｗｅｌｌ　Ｐｌａｔｅ（コーニング社製）において、５％ＣＯ２、３７℃の条件で培養した。 OV90, OVCAR3, SKOV3, and Ishikawa were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 5% CO 2 , 37 ° C.
A2780 and RMG1 were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 5% CO 2 , 37 ° C.
ES2 was cultured in DMEM / F12 medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin under the conditions of 5% CO 2 and 37 ° C.
BrC-PDC is 8.8 ng / mL basic fibroblast growth factor (Thermo Fisher Scientific), 20 μmol / LY-27632 (Rho-associated coiled-coil forming kinase inhibitor), Thermo Fisher. , 50 U / mL penicillin, 50 μg / mL streptomycin in StemPro hESC SFM medium (Thermo Fisher Scientific) in Ultra-Low Attachment Multiple Well Plate (Corning) at 5% CO 2 , 37 ° C. It was cultured under the conditions.

＜＜ｓｉＲＮＡ及びプラスミドのトランスフェクション＞＞
　ＯＩＮ１に対する二本鎖核酸分子として、ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｅｎｃｅ　ＲＮＡ（ｓｉＲＮＡ）を設計し、「ｓｉＯＩＮ１　＃１」、「ｓｉＯＩＮ１　＃２」と命名して、シグマアルドリッチジャパン社にて合成した。
　また、コントロールとして使用した二本鎖核酸分子（ｓｉＣｏｎｔｒｏｌ）は、ＲＮＡｉ社より購入した。
　以下に、各ｓｉＲＮＡの配列を示す。 << Transfection of siRNA and plasmid >>
Small interference RNA (siRNA) was designed as a double-stranded nucleic acid molecule for OIN1, named "siOIN1 # 1" and "siOIN1 # 2", and synthesized by Sigma-Aldrich Japan.
The double-stranded nucleic acid molecule (siControl) used as a control was purchased from RNAi.
The sequence of each siRNA is shown below.

［ｓｉＯＩＮ１　＃１］
－標的配列－
　５’－ＧＣＴＣＡＧＣＴＣＡＣＧＧＣＴＴＣＴＡＣＣ－３’（配列番号：２）
－二本鎖核酸分子（ｓｉＲＮＡ）の配列－
　・　センス鎖
　　　５’－ＧＣＵＣＡＧＣＵＣＡＣＧＧＣＵＵＣＵＡＣＣ－３’（配列番号：４）
　・　アンチセンス鎖
　　　５’－ＵＡＧＡＡＧＣＣＧＵＧＡＧＣＵＧＡＧＣＵＣ－３’（配列番号：５） [SiOIN1 # 1]
-Target sequence-
5'-GCTCAGCTCACGGCTTCTAC-3'(SEQ ID NO: 2)
-Sequence of double-stranded nucleic acid molecule (siRNA)-
-Sense strand 5'-GCUCAGCUCACGCGCUUCUC-3'(SEQ ID NO: 4)
-Antisense strand 5'-UAGAAGCCGUGAGCUGAGCUC-3'(SEQ ID NO: 5)

［ｓｉＯＩＮ１　＃２］
－標的配列－
　５’－ＧＡＣＡＧＧＡＧＡＣＴＣＣＡＧＡＡＡＡＧＧ－３’（配列番号：３）
－二本鎖核酸分子（ｓｉＲＮＡ）の配列－
　・　センス鎖
　　　５’－ＧＡＣＡＧＧＡＧＡＣＵＣＣＡＧＡＡＡＡＧＧ－３’（配列番号：６）
　・　アンチセンス鎖
　　　５’－ＵＵＵＵＣＵＧＧＡＧＵＣＵＣＣＵＧＵＣＵＧ－３’（配列番号：７） [SiOIN1 # 2]
-Target sequence-
5'-GACAGGAGACTCCAGAAAAGG-3'(SEQ ID NO: 3)
-Sequence of double-stranded nucleic acid molecule (siRNA)-
-Sense strand 5'-GACAGGAGACUCCAGAAAGG-3'(SEQ ID NO: 6)
-Antisense strand 5'-UUUUCUGGAGUCUCCUGUCUG-3'(SEQ ID NO: 7)

［ｓｉＣｏｎｔｒｏｌ］
－二本鎖核酸分子（ｓｉＲＮＡ）の配列－
　・　センス鎖
　　　５’－ＧＵＡＣＣＧＣＡＣＧＵＣＡＵＵＣＧＵＡＵＣ－３’（配列番号：８）
　・　アンチセンス鎖
　　　５’－ＧＡＵＡＣＧＡＡＵＧＡＣＧＵＧＣＧＧＵＡＣ－３’（配列番号：９） [SiControl]
-Sequence of double-stranded nucleic acid molecule (siRNA)-
-Sense strand 5'-GUACCGCACGUCAUCGUAUC-3'(SEQ ID NO: 8)
-Antisense strand 5'-GAUACGAAUGACGUGCGGUAC-3'(SEQ ID NO: 9)

　前記ｓｉＲＮＡ又はＯＩＮ１遺伝子を発現するプラスミドをトランスフェクションする前に、Ａ２７８０及びＲＭＧ１の場合は３×１０５細胞ずつ、ＳＫＯＶ３の場合は１×１０５細胞ずつ、細胞を６ウェルプレートのウェルに播いた。
　２４時間培養後に、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭａｘ（サーモフィッシャーサイエンティフィック社製）を使用して、前記ｓｉＲＮＡを終濃度が１０ｎＭとなるように細胞にトランスフェクションした。ＯＩＮ１遺伝子を発現するプラスミド又は空のベクターのトランスフェクションはＦｕＧｅｎｅ　ＨＤ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（プロメガ社製）を用いて行なった。なお、トランスフェクションの方法は、使用した製品のプロトコルに従った。
　トランスフェクションの４８時間又は７２時間後に細胞を回収して、後述する定量的ＲＴ－ＰＣＲ実験に使用した。 Prior to transfection of the plasmid expressing the siRNA or OIN1 gene, cells were seeded in wells of a 6-well plate with 3 × 10 5 cells each for A2780 and RMG1 and 1 × 10 5 cells each for SKOV3. ..
After culturing for 24 hours, the siRNA was transfected into cells to a final concentration of 10 nM using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific). Transfection of a plasmid expressing the OIN1 gene or an empty vector was performed using FuGene HD Transfection Reagent (manufactured by Promega). The transfection method followed the protocol of the product used.
Cells were harvested 48 or 72 hours after transfection and used in the quantitative RT-PCR experiments described below.

＜＜ＲＮＡの抽出と定量的ＲＴ－ＰＣＲ（ｑＲＴ－ＰＣＲ）＞＞
　ＩＳＯＧＥＮ試薬（株式会社ニッポンジーン製）を使用して、卵巣がん細胞、又はＡ２７８０由来の異種移植腫瘍からＲＮＡを抽出した。なお、腫瘍の破砕は、Ｐｏｌｙｔｒｏｎ　ＰＴ３１００（Ｋｉｎｅｍａｔｉｃａ社製）を使用した。
　ＳｕｐｅｒＳｃｒｉｐｔ　ＩＩＩ（サーモフィッシャーサイエンティフィック社製）とランダムヘキサマープライマーを使用して、１μｇの抽出ＲＮＡから一本鎖ｃＤＮＡを逆転写により合成した。
　ｑＲＴ－ＰＣＲは、作製したｃＤＮＡとＫＡＰＡ　ＳＹＢＲ　ＦＡＳＴ　ｑＰＣＲキット（ＫＡＰＡ　Ｂｉｏｓｙｓｔｅｍｓ社製）、及び遺伝子に特異的なプライマーのセットを使用して、ＳｔｅｐＯｎｅＰｌｕｓ　Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ　Ｓｙｓｔｅｍ（サーモフィッシャーサイエンティフィック社製）で行った。
　ＳｔｅｐＯｎｅＰｌｕｓ　Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ　Ｓｙｓｔｅｍの製品のプロトコルに従ってΔΔＣｔ法によってＲＮＡの発現量を分析し、ＧＡＰＤＨ遺伝子の発現量を補正に用いた。
　ｑＲＴ－ＰＣＲに使用したプライマーは、下記のとおりである。 << RNA extraction and quantitative RT-PCR (qRT-PCR) >>
RNA was extracted from ovarian cancer cells or xenograft tumors derived from A2780 using ISOGEN reagent (manufactured by Nippon Gene Co., Ltd.). For crushing the tumor, Polytron PT3100 (manufactured by Kinematica) was used.
Single-stranded cDNA was synthesized by reverse transcription from 1 μg of extracted RNA using SuperScript III (manufactured by Thermo Fisher Scientific) and a random hexamer primer.
qRT-PCR uses the prepared cDNA, the KAPA SYBR FAST qPCR kit (manufactured by KAPA Biosystems), and a set of gene-specific primers to be used in the StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific). I went there.
RNA expression was analyzed by the ΔΔCt method according to the StepOnePlus Real-Time PCR System product protocol, and the GAPDH gene expression was used for correction.
The primers used for qRT-PCR are as follows.

［ＧＡＰＤＨ］
　・　フォワード
　　　５’－ＧＧＴＧＧＴＣＴＣＣＴＣＴＧＡＣＴＴＣＡＡＣＡ－３’（配列番号：１０）
　・　リバース
　　　５’－ＧＴＧＧＴＣＧＴＴＧＡＧＧＧＣＡＡＴＧ－３’（配列番号：１１）
［ＯＩＮ１］
　・　フォワード
　　　５’－ＴＣＴＴＣＡＣＣＣＣＴＡＡＣＣＡＧＣＡＧＧＡＡ－３’（配列番号：１２）
　・　リバース
　　　５’－ＡＧＧＡＣＴＧＡＡＧＴＡＡＧＴＣＣＴＧＡＴＧＣ－３’（配列番号：１３）
［ＭＣＭ５］
　・　フォワード
　　　５’－ＡＧＣＡＴＴＣＧＴＡＧＣＣＴＧＡＡＧＴＣＧ－３’（配列番号：１４）
　・　リバース
　　　５’－ＣＧＧＣＡＣＴＧＧＡＴＡＧＡＧＡＴＧＣＧ－３’（配列番号：１５）
［Ｅ２Ｆ３］
　・　フォワード
　　　５’－ＡＧＡＡＡＧＣＧＧＴＣＡＴＣＡＧＴＡＣＣＴ－３’（配列番号：１６）
　・　リバース
　　　５’－ＴＧＧＡＣＴＴＣＧＴＡＧＴＧＣＡＧＣＴＣＴ－３’（配列番号：１７）
［ＰＩＫ３ＣＢ］
　・　フォワード
　　　５’－ＣＴＧＣＣＴＧＣＧＡＣＡＧＡＴＧＡＧＴＧ－３’（配列番号：１８）
　・　リバース
　　　５’－ＴＣＣＧＡＴＴＡＣＣＡＡＧＴＧＣＴＣＴＴＴＣ－３’（配列番号：１９）
［ＮＯＴＣＨ１］
　・　フォワード
　　　５’－ＧＡＧＧＣＧＴＧＧＣＡＧＡＣＴＡＴＧＣ－３’（配列番号：２０）
　・　リバース
　　　５’－ＣＴＴＧＴＡＣＴＣＣＧＴＣＡＧＣＧＴＧＡ－３’（配列番号：２１）
［ＣＤＫＮ１Ｂ］
　・　フォワード
　　　５’－ＴＡＡＴＴＧＧＧＧＣＴＣＣＧＧＣＴＡＡＣＴ－３’（配列番号：２２）
　・　リバース
　　　５’－ＴＧＣＡＧＧＴＣＧＣＴＴＣＣＴＴＡＴＴＣＣ－３’（配列番号：２３）
［ＲＡＳＳＦ５］
　・　フォワード
　　　５’－ＧＧＧＣＡＴＧＡＡＡＣＴＧＡＧＴＧＡＡＧＡ－３’（配列番号：２４）
　・　リバース
　　　５’－ＴＧＧＣＡＴＣＡＴＡＧＡＴＧＧＡＣＴＧＧＧ－３’（配列番号：２５）
［ＡＤＯＲＡ１］
　・　フォワード
　　　５’－ＣＣＡＣＡＧＡＣＣＴＡＣＴＴＣＣＡＣＡＣＣ－３’（配列番号：２６）
　・　リバース
　　　５’－ＴＡＣＣＧＧＡＧＡＧＧＧＡＴＣＴＴＧＡＣＣ－３’（配列番号：２７）
［ＲＢＭ５］
　・　フォワード
　　　５’－ＡＴＧＧＧＴＴＣＡＧＡＣＡＡＡＡＧＡＧＴＧＡＧ－３’（配列番号：２８）
　・　リバース
　　　５’－ＣＴＧＣＴＴＣＧＧＧＡＴＴＣＡＣＧＣＴ－３’（配列番号：２９）
［ＲＢＭ６］
　・　フォワード
　　　５’－ＴＧＧＡＧＴＡＴＧＴＡＴＣＡＡＧＣＣＴＧＧＡ－３’（配列番号：３０）
　・　リバース
　　　５’－ＡＴＧＡＡＣＡＧＧＡＡＧＡＴＣＧＧＴＧＣＣ－３’（配列番号：３１） [GAPDH]
-Forward 5'-GGTGGTCTCCCTTGACTCAACA-3'(SEQ ID NO: 10)
-Reverse 5'-GTGGTCGTTGGGCAATG-3'(SEQ ID NO: 11)
[OIN1]
-Forward 5'-TCTTCACCCCTAACCAGCAGGAA-3' (SEQ ID NO: 12)
-Reverse 5'-AGGACTGAAGTAAGTCCGATGC-3'(SEQ ID NO: 13)
[MCM5]
-Forward 5'-AGCATTCGTAGCCTGAAGTCG-3'(SEQ ID NO: 14)
-Reverse 5'-CGGCACTGGATAGAGAGCG-3'(SEQ ID NO: 15)
[E2F3]
-Forward 5'-AGAAAGCGGTCATCAGTACT-3'(SEQ ID NO: 16)
-Reverse 5'-TGGACTTCGTAGTGCAGCTCT-3'(SEQ ID NO: 17)
[PIK3CB]
-Forward 5'-CTGCCTGCGACAGATTGAGTG-3'(SEQ ID NO: 18)
-Reverse 5'-TCCGATTACCAAGTGCTCTTC-3'(SEQ ID NO: 19)
[NOTCH1]
-Forward 5'-GAGGCGTGGGCAGACTATGC-3'(SEQ ID NO: 20)
-Reverse 5'-CTTGTACCTCCGTCAGCGTGA-3' (SEQ ID NO: 21)
[CDKN1B]
-Forward 5'-TAATTGGGGCTCCGGCTAACT-3'(SEQ ID NO: 22)
-Reverse 5'-TGCAGGTCCGCTTCCTATTCC-3'(SEQ ID NO: 23)
[RASSF5]
-Forward 5'-GGGCATGAAACTGAGTGAAGA-3'(SEQ ID NO: 24)
-Reverse 5'-TGGCATCATCATGGACTGG-3'(SEQ ID NO: 25)
[ADORA1]
-Forward 5'-CCACAGACCACTACTTCACCACC-3'(SEQ ID NO: 26)
-Reverse 5'-TACCGGAGAGGGATCTTGACC-3'(SEQ ID NO: 27)
[RBM5]
-Forward 5'-ATGGGTTCAGACAAAAGAGTGAG-3'(SEQ ID NO: 28)
-Reverse 5'-CTGCTTCGGGATTCACGCT-3'(SEQ ID NO: 29)
[RBM6]
-Forward 5'-TGGAGTATGTATCAAGCCTGGA-3'(SEQ ID NO: 30)
-Reverse 5'-ATGAACAGGAAGATCGGTGCC-3'(SEQ ID NO: 31)

＜＜細胞増殖の解析＞＞
　Ａ２７８０及びＲＭＧ１の場合は３，０００細胞ずつ、ＳＫＯＶ３の場合は１，０００細胞ずつ、Ｉｓｈｉｋａｗａの場合は２，０００細胞ずつ、細胞を９６ウェルプレートのウェルに播いた。
　２４時間培養した後、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭａｘ（サーモフィッシャーサイエンティフィック社製）を使用して、ｓｉＲＮＡを終濃度が１０ｎＭとなるように細胞にトランスフェクションした。細胞は播いてから１日後、３日後、５日後に回収した。ＯＩＮ１遺伝子を発現するプラスミド又は空のベクターのトランスフェクションは、ＦｕＧｅｎｅ　ＨＤ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（プロメガ社製）を使用して行った。細胞は播いてから１日後、３日後に回収した。
　細胞増殖能について評価するために、ウェル内の細胞のＤＮＡをＨｏｅｃｈｓｔ　３３２５８　ｐｅｎｔａｈｙｄｒａｔｅ（サーモフィッシャーサイエンティフィック社製、終濃度５μｇ／ｍＬ）で染色した。各ウェルのＤＮＡ量を２０３０　ＡＲＶＯ　Ｘ５　Ｍｕｌｔｉｌａｂｅｌ　Ｐｌａｔｅ　Ｒｅａｄｅｒ（パーキンエルマー社）により測定した（ＤＮＡアッセイ）。 << Analysis of cell proliferation >>
Cells were seeded in 96-well plate wells, 3,000 cells each for A2780 and RMG1, 1,000 cells each for SKOV3, and 2,000 cells each for Ishikawa.
After culturing for 24 hours, cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific). The cells were collected 1 day, 3 days and 5 days after seeding. Transfection of a plasmid expressing the OIN1 gene or an empty vector was performed using FuGene HD Transfection Reagent (manufactured by Promega). The cells were collected 1 day and 3 days after seeding.
To evaluate cell proliferation, the DNA of the cells in the wells was stained with Hoechst 33258 pentahydrate (Thermo Fisher Scientific, final concentration 5 μg / mL). The amount of DNA in each well was measured by a 2030 ARVO X5 Multilabel Plate Reader (PerkinElmer) (DNA assay).

　ＢｒＣ－ＰＤＣの場合は、４，００００細胞ずつ２４０μＬのＯｐｔｉ－ＭＥＭ培地（サーモフィッシャーサイエンティフィック社製）に懸濁して、２４ウェルプレート（Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ　Ｍｕｌｔｉｐｌｅ　Ｗｅｌｌ　Ｐｌａｔｅ、コーニング社製）に播いた。その直後、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭａｘ（サーモフィッシャーサイエンティフィック社製）を使用して、ｓｉＲＮＡを終濃度が１０ｎＭとなるように細胞にトランスフェクションした。トランスフェクションから６時間後にＳｔｅｍＰｒｏ　ｈＥＳＣ　ＳＦＭ培地（サーモフィッシャーサイエンティフィック社製）を２００μＬ加えた。細胞は播いてから５日後に回収し、さらに各ウェルの細胞を９６ウェルプレートの５ウェルに等分して播いた。
　その後、細胞増殖能を評価するために、ウェル内の細胞のＡＴＰ量をＣｅｌｌＴｉｔｅｒ－Ｇｌｏ　３Ｄ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ　Ａｓｓａｙ（プロメガ社製）及びＴｒｉＳｔａｒ２　Ｓ　ＬＢ　９４２　Ｍｕｌｔｉｍｏｄｅ　Ｒｅａｄｅｒ（Ｂｅｒｔｈｏｌｄ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）を用いて測定した。 In the case of BrC-PDC, 4,000 cells are suspended in 240 μL of Opti-MEM medium (manufactured by Thermo Fisher Scientific) and seeded on a 24-well plate (Ultra-Low Attachment Multiple Well Plate, manufactured by Corning). board. Immediately thereafter, cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific). Six hours after transfection, 200 μL of StemPro hESC SFM medium (manufactured by Thermo Fisher Scientific) was added. The cells were collected 5 days after seeding, and the cells in each well were further divided into 5 wells of a 96-well plate and seeded.
Then, in order to evaluate the cell proliferation ability, the amount of ATP of the cells in the well was measured using CellTiter - Glo 3D Cell Viability Assay (manufactured by Promega) and TriStar 2S LB 942 Multimode Reader (manufactured by Berthold Technologies). ..

＜＜アネキシンＶとヨウ化プロピジウム（ＰＩ）を使用したアポトーシスの解析＞＞
　Ａ２７８０の場合は３×１０５細胞ずつ、ＳＫＯＶ３の場合は１×１０５細胞ずつ、６ウェルプレートのウェルに播いた。２４時間培養した後、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭａｘを使用して、ｓｉＲＮＡを終濃度が１０ｎＭとなるように細胞にトランスフェクションし、７２時間後に細胞を回収した。
　アポトーシスを起こした細胞は、ＦＩＴＣ　Ａｎｎｅｘｉｎ　Ｖ　Ａｐｏｐｔｏｓｉｓ　Ｄｅｔｅｃｔｉｏｎ　Ｋｉｔ　Ｉ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用いて製品のプロトコルに従い染色した。アネキシンＶとＰＩで染色される細胞をＢＤ　ＦＡＣＳＣａｌｉｂｕｒ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）により解析した。 << Analysis of Apoptosis Using Annexin V and Propidium Iodide (PI) >>
In the case of A2780, 3 × 10 5 cells were seeded, and in the case of SKOV3, 1 × 10 5 cells were seeded in the wells of a 6-well plate. After culturing for 24 hours, cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax, and the cells were harvested after 72 hours.
Apopulated cells were stained with FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the product protocol. Cells stained with Annexin V and PI were analyzed by BD FACSCalibur (BD Biosciences).

＜＜Ｉｎ　ｖｉｖｏでの腫瘍形成実験＞＞
　全ての動物実験は埼玉医科大学動物実験委員会の承認の下、埼玉医科大学動物実験規程に従い行われた。雌のヌードマウス（ＢＡＬＢ／ｃＡＪｃＩ－ｎｕ／ｎｕ）は日本クレア株式会社より購入した。Ａ２７８０（１×１０５細胞）と等量のＭａｔｒｉｇｅｌ　ｍａｔｒｉｘ（コーニング社製）を混合して、１０週齢の雌のヌードマウスの脇腹に皮下注射した。その後、マウスをランダムに２群に分けた。ｓｉＣｏｎｔｒｏｌ又はｓｉＯＩＮ１　＃１（それぞれ５μｇ）をトランスフェクション試薬であるＧｅｎｅＳｉｌｅｎｃｅｒ　ｒｅａｇｅｎｔ（Ｇｅｎｅ　Ｔｈｅｒａｐｙ　Ｓｙｓｔｅｍ社製）と混合して、週に２回、マウスに形成された腫瘍に注射した。
　腫瘍体積の測定は週に一度行い、０．５×（１軸目の直径）×（２軸目の直径）×（３軸目の直径）という式により算出した。 << Tumor formation experiment in vivo >>
All animal experiments were conducted in accordance with the Saitama Medical University Animal Experiment Regulations under the approval of the Saitama Medical University Animal Experiment Committee. Female nude mice (BALB / cAJcI-nu / nu) were purchased from Japan Claire Co., Ltd. A2780 (1 × 10 5 cells) and an equal amount of Matrix matrix (manufactured by Corning) were mixed and subcutaneously injected into the flank of a 10-week-old female nude mouse. Then, the mice were randomly divided into two groups. SiControl or siOIN1 # 1 (5 μg each) was mixed with the transfection reagent GeneSilicer reagent (manufactured by Gene Therapy System) and injected into the tumor formed in mice twice a week.
The tumor volume was measured once a week and calculated by the formula of 0.5 × (diameter of the first axis) × (diameter of the second axis) × (diameter of the third axis).

＜＜統計解析＞＞
　データの統計解析は、Ｍａｎｎ－ＷｈｉｔｎｅｙのＵ検定、２元配置分散分析（ｔｗｏ－ｗａｙ　ａｎａｌｙｓｉｓ　ｏｆ　ｖａｒｉａｎｃｅ；　ｔｗｏ－ｗａｙ　ＡＮＯＶＡ）、又はＳｔｕｄｅｎｔのｔ検定により行った。どの検定手法を用いたかに関しては各データにおいて示した。統計解析のためソフトウェアとして、Ｍｉｃｒｏｓｏｆｔ　Ｅｘｃｅｌ（マイクロソフト社製）とＪＭＰ　９．０．００（ＳＡＳ　Ｉｎｓｔｉｔｕｔｅ社製）を使用した。 << Statistical analysis >>
Statistical analysis of the data was performed by Mann-Whitney U-test, two-way analysis of variance (two-way ANOVA), or Student's t-test. Which test method was used is shown in each data. For statistical analysis, Microsoft Excel (manufactured by Microsoft) and JMP 9.0.00 (manufactured by SAS Institute) were used as software.

＜結果＞
＜＜卵巣がん細胞においてｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｌｏｎｇ　ｉｎｔｅｒｇｅｎｉｃ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡ　１（ＯＩＮ１）は高発現する＞＞
　卵巣がんで特異的に発現する長鎖非コードＲＮＡを探索するために、卵巣の正常組織（Ｎｏｒｍａｌ、ｎ＝６）と、卵巣明細胞がん（ＯＣＣＣ、ｎ＝６）及び高異型度漿液性卵巣がん（ＨＧＳＯＣ、ｎ＝１５）の臨床検体を使用したＲＮＡシーケンシング解析を行った。
　その結果、ＮＯＮＣＯＤＥデータベース（ＮＯＮＣＯＤＥ．　Ａｖａｉｌａｂｌｅ　ｏｎｌｉｎｅ：　ｈｔｔｐ：／／ｗｗｗ．ｎｏｎｃｏｄｅ．ｏｒｇ／　（ａｃｃｅｓｓｅｄ　ｏｎ　３１　Ｍａｙ　２０２０）．）においてＮＯＮＨＳＡＴ０１３４４８として登録された長鎖非コードＲＮＡが、卵巣の正常組織と比較して、卵巣明細胞がんと高異型度漿液性卵巣がんにおいて高発現することが明らかとなった（図１Ａ及びＢ参照）。 <Result>
<< Ovarian cancer cells are highly expressed in ovarian cancer intergenic noncoding RNA 1 (OIN1) >>
To search for long non-coding RNA specifically expressed in ovarian cancer, normal ovarian tissue (Normal, n = 6), ovarian clear cell carcinoma (OCCC, n = 6) and high-grade serous carcinoma. RNA sequencing analysis was performed using clinical specimens of ovarian cancer (HGSOC, n = 15).
As a result, the long non-coding RNA registered as NONHSAT013448 in the NONCODE database (NONCODE. Archive online: http: //www.noncode.org/ (accessed on 31 May 2020)) was compared with the normal tissue of the ovary. It was revealed that it is highly expressed in clear cell ovarian cancer and highly atypical serous ovarian cancer (see FIGS. 1A and 1B).

　図１Ａは、卵巣の正常組織、卵巣明細胞がん、及び高異型度漿液性卵巣がんにおけるＮＯＮＨＳＡＴ０１３４４８として登録された長鎖非コードＲＮＡ（ＯＩＮ１）の発現量を比較した結果を示す図である。ＮＯＮＨＳＡＴ０１３４４８として登録された長鎖非コードＲＮＡは、正常組織と比較して卵巣明細胞がん及び高異型度漿液性卵巣がんで発現量が高かった。ＮＯＮＨＳＡＴ０１３４４８として登録された長鎖非コードＲＮＡの発現量はＲＰＫＭ（ｒｅａｄｓ　ｐｅｒ　ｋｉｌｏｂａｓｅ　ｐｅｒ　ｍｉｌｌｉｏｎ　ｓｅｑｕｅｎｃｅｄ　ｒｅａｄｓ）の値から算出した（Ｊ．Ｙ．　Ｗａｎｇ，　Ａ．Ｑ．　Ｌｕ，　Ｌ．Ｊ．　Ｃｈｅｎ，　ＬｎｃＲＮＡｓ　ｉｎ　ｏｖａｒｉａｎ　ｃａｎｃｅｒ，　Ｃｌｉｎ．　Ｃｈｉｍ．　Ａｃｔａ，　４９０　（２０１９）　１７－２７，　ｄｏｉ：　１０．１０１６／ｊ．ｃｃａ．２０１８．１２．０１３．）。図１Ａ中、＊はｐ＜０．０５、＊＊はｐ＜０．０１を表す。統計解析はＭａｎｎ－ＷｈｉｔｎｅｙのＵ検定により行った。 FIG. 1A is a diagram showing the results of comparing the expression levels of long non-coding RNA (OIN1) registered as NONHSAT013448 in normal ovarian tissue, clear ovarian cell cancer, and highly atypical serous ovarian cancer. .. The long non-coding RNA registered as NONHSAT013448 was higher in expression in clear ovarian cell carcinoma and highly atypical serous ovarian cancer compared to normal tissue. The expression level of the long-chain non-coding RNA registered as NONHSAT013448 was calculated from the value of RPKM (reads per kilobase per million defined reads) (JY Wang, AQ Lu, LJ inLn, Inc.). ovarian cancer, Clin. Chim. Acta, 490 (2019) 17-27, doi: 10.1016 / j.cca.2018.12.013.). In FIG. 1A, * represents p <0.05 and ** represents p <0.01. Statistical analysis was performed by the Mann-Whitney U test.

　ＮＯＮＨＳＡＴ０１３４４８として登録された長鎖非コードＲＮＡは、同じくＮＯＮＣＯＤＥデータベースにおいてＮＯＮＨＳＡＧ００５９３０として登録された遺伝子より転写される。ＮＯＮＨＳＡＧ００５９３０遺伝子はヒトの１０番染色体の長腕に存在し（１０ｑ２１．１）、上流と下流で最も近くに存在するタンパク質をコードする遺伝子であるｐｒｏｔｏｃａｄｈｅｒｉｎ－ｒｅｌａｔｅｄ　１５（ＰＣＤＨ１５）とｍａｎｎｏｓｅ　ｂｉｎｄｉｎｇ　ｌｅｃｔｉｎ　２（ＭＢＬ２）とは、それぞれ約０．７８Ｍｂまた約０．２０Ｍｂ離れている。すなわち、ＮＯＮＨＳＡＧ００５９３０遺伝子はタンパク質をコードする遺伝子と重なる領域を持たないことから、ＮＯＮＨＳＡＴ０１３４４８は長鎖非コードＲＮＡの中でもｌｏｎｇ　ｉｎｔｅｒｇｅｎｉｃ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡというカテゴリーに分類される。 The long non-coding RNA registered as NONHSAT013448 is transcribed from the gene also registered as NONHSAG005930 in the NONCODE database. The NONSAG005930 gene is present in the long arm of human chromosome 10 (10q21.1), and is a gene encoding a protein that is closest to upstream and downstream, protocadherin-related 15 (PCDH15) and mannose binding lectin 2 (MBL2). ) Are separated from each other by about 0.78 Mb and about 0.20 Mb, respectively. That is, since the NONHSAG005930 gene does not have a region overlapping with the gene encoding the protein, NONHSAT013448 is classified into the long intergenic noncoding RNA category among the long non-coding RNAs.

　ＮＯＮＨＳＡＴ０１３４４８の機能は、これまで研究がなされておらず不明であった。そこで、この長鎖非コードＲＮＡをｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｌｏｎｇ　ｉｎｔｅｒｇｅｎｉｃ　ｎｏｎｃｏｄｉｎｇ　ＲＮＡ　１（ＯＩＮ１）と命名した。なお、ＯＩＮ１の塩基配列は、配列番号：１のとおりであった。 The function of NONHSAT013448 has not been studied so far and was unknown. Therefore, this long non-coding RNA was named ovarian cancer long intergenic noncoding RNA 1 (OIN1). The base sequence of OIN1 was as shown in SEQ ID NO: 1.

　図１Ｂの上図は、卵巣の正常組織と、卵巣明細胞がん及び高異型度漿液性卵巣がんの検体に由来するＲＮＡシーケンシングのリードをＯＩＮ１の遺伝子座へマッピングした図であり、下図は、ＯＩＮ１は２つのエクソンにより構成されることを示した図である。 The upper figure of FIG. 1B is a diagram in which normal tissue of the ovary and RNA-seqing leads derived from specimens of clear cell ovarian cancer and highly atypical serous ovarian cancer are mapped to the locus of OIN1. Is a diagram showing that OIN1 is composed of two exons.

　次に、卵巣がん細胞におけるＯＩＮ１の発現をｑＲＴ－ＰＣＲ法により調べた。 Next, the expression of OIN1 in ovarian cancer cells was examined by the qRT-PCR method.

　図１Ｃは、卵巣がん細胞におけるＯＩＮ１の発現量をｑＲＴ－ＰＣＲ法により解析し、ＧＡＰＤＨ　ｍＲＮＡの発現量で補正した結果を示す図である。図１Ｃでは、ＯＩＮ１の発現量の平均値±ＳＤを示している（ｎ＝３）。 FIG. 1C is a diagram showing the results of analyzing the expression level of OIN1 in ovarian cancer cells by the qRT-PCR method and correcting for the expression level of GAPDH mRNA. FIG. 1C shows the average value ± SD of the expression level of OIN1 (n = 3).

　図１Ｃに示したように、ＯＩＮ１はＡ２７８０、ＳＫＯＶ３、及びＲＭＧ１において高発現し、ＯＶ９０細胞において弱く発現することが明らかとなった（図１Ｃ参照）。
　ＯＩＮ１を高発現することから、Ａ２７８０、ＳＫＯＶ３、及びＲＭＧ１をＯＩＮ１の機能解析実験に用いることとした。 As shown in FIG. 1C, OIN1 was found to be highly expressed in A2780, SKOV3, and RMG1 and weakly expressed in OV90 cells (see FIG. 1C).
Since OIN1 is highly expressed, A2780, SKOV3, and RMG1 were used for the functional analysis experiment of OIN1.

＜＜ＯＩＮ１は卵巣がん細胞の増殖を促進し、アポトーシスを抑制する＞＞
　次に卵巣がんにおけるＯＩＮ１の役割を探るために、ｓｉＲＮＡ（ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２）を使用したＯＩＮ１のノックダウン実験を行った。 << OIN1 promotes the growth of ovarian cancer cells and suppresses apoptosis >>
Next, in order to explore the role of OIN1 in ovarian cancer, a knockdown experiment of OIN1 using siRNA (siOIN1 # 1 and siOIN1 # 2) was performed.

　図２Ａは、ＯＩＮ１に対するｓｉＲＮＡ（ｓｉＯＩＮ１　＃１とｓｉＯＩＮ１　＃２）によるＯＩＮ１のノックダウン効率をＡ２７８０（図２Ａの左）、ＳＫＯＶ３（図２Ａの中央）、ＲＭＧ１（図２Ａの右）でｑＲＴ－ＰＣＲにより解析した結果を示す図である。ＯＩＮ１の相対的な発現量をＧＡＰＤＨ　ｍＲＮＡの発現量で補正して算出し、図２Ａでは、ｓｉＣｏｎｔｒｏｌを処理した条件と比較したときのＯＩＮ１の発現倍率の変化の平均値±ＳＤを示している（ｎ＝３）。図２Ａ中、＊＊＊はｐ＜０．０００１を表す。統計解析は２元配置分散分析により行った。 FIG. 2A shows the knockdown efficiency of OIN1 by siRNA (siOIN1 # 1 and siOIN1 # 2) with respect to OIN1 in A2780 (left in FIG. 2A), SKOV3 (center in FIG. 2A), and qRT-PCR in RMG1 (right in FIG. 2A). It is a figure which shows the result of the analysis by. The relative expression level of OIN1 was corrected by the expression level of GAPDH mRNA and calculated, and FIG. 2A shows the average value ± SD of the change in the expression ratio of OIN1 when compared with the condition treated with siControl ( n = 3). In FIG. 2A, *** represents p <0.0001. Statistical analysis was performed by two-way ANOVA.

　図２Ａに示したように、ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２の処理がＡ２７８０、ＳＫＯＶ３、及びＲＭＧ１においてＯＩＮ１の発現を有意に減少させることが明らかとなった。 As shown in FIG. 2A, it was revealed that the treatment of siOIN1 # 1 and siOIN1 # 2 significantly reduced the expression of OIN1 in A2780, SKOV3, and RMG1.

　さらに、ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２によるＯＩＮ１のノックダウンが、これらの卵巣がん細胞の増殖を有意に抑制することをＤＮＡアッセイにより確認した。結果を図２Ｂに示す。 Furthermore, it was confirmed by DNA assay that knockdown of OIN1 by siOIN1 # 1 and siOIN1 # 2 significantly suppressed the growth of these ovarian cancer cells. The results are shown in FIG. 2B.

　図２Ｂ中、左はＡ２７８０、中央はＳＫＯＶ３、右はＲＭＧ１の結果を示す。図２ＢではＤＮＡ量の測定値の平均値±ＳＤを示している（Ａ２７８０はｎ＝５、ＳＫＯＶ３はｎ＝３、ＲＭＧ１はｎ＝３）。図２Ｂ中、＊＊はｐ＜０．００１、＊＊＊はｐ＜０．０００１を表す。統計解析は２元配置分散分析により行った。 In FIG. 2B, the left is A2780, the center is SKOV3, and the right is RMG1. FIG. 2B shows the average value ± SD of the measured values of the amount of DNA (A2780 is n = 5, SKOV3 is n = 3, RMG1 is n = 3). In FIG. 2B, ** represents p <0.001 and *** represents p <0.0001. Statistical analysis was performed by two-way ANOVA.

　これらの結果はＯＩＮ１が卵巣がんの増殖に重要な役割を果たすことを示している。 These results indicate that OIN1 plays an important role in the growth of ovarian cancer.

　次に、ＯＩＮ１がどのようにして卵巣がんの増殖を制御するかに関して明らかにするために、ＯＩＮ１のノックダウンが卵巣がん細胞のアポトーシスに与える影響を調べた。 Next, in order to clarify how OIN1 regulates the growth of ovarian cancer, the effect of knockdown of OIN1 on the apoptosis of ovarian cancer cells was investigated.

　ＰＩ及びアネキシンＶによる細胞染色とフローサイトメトリーを組み合わせた手法によりアポトーシスを解析した結果を図２Ｃ～２Ｆに示す。 Figures 2C-2F show the results of analysis of apoptosis by a method that combines cell staining with PI and Annexin V and flow cytometry.

　細胞としてＡ２７８０を用いた場合の結果を図２Ｃ及び２Ｅに示し、細胞としてＳＫＯＶ３を用いた場合の結果を図２Ｄ及び２Ｆに示す。図２Ｃ及び２Ｄに示したように、ＯＩＮ１のノックダウンがＡ２７８０とＳＫＯＶ３のアポトーシスを促進することがＰＩとアネキシンＶによる細胞染色及びフローサイトメトリーにより明らかとなった。また、図２Ｅ及び２Ｆでは、Ａ２７８０とＳＫＯＶ３でアポトーシスを起こした細胞の割合を定量してグラフに示した。図２Ｅ及び２Ｆ中、＊＊はｐ＜０．００１、＊＊＊はｐ＜０．０００１を表す。統計解析は２元配置分散分析により行った。 The results when A2780 is used as cells are shown in FIGS. 2C and 2E, and the results when SKOV3 is used as cells are shown in FIGS. 2D and 2F. As shown in FIGS. 2C and 2D, knockdown of OIN1 promotes apoptosis of A2780 and SKOV3 by cell staining and flow cytometry with PI and Annexin V. In addition, in FIGS. 2E and 2F, the proportion of cells that have undergone apoptosis in A2780 and SKOV3 is quantified and shown in a graph. In FIGS. 2E and 2F, ** represents p <0.001 and *** represents p <0.0001. Statistical analysis was performed by two-way ANOVA.

　以上のように、ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２は、Ａ２７８０とＳＫＯＶ３のアポトーシスを増加させることが示された。そのため、ＯＩＮ１はアポトーシスの抑制を介して卵巣がん細胞の増殖を促進することが示された。 As described above, it was shown that siOIN1 # 1 and siOIN1 # 2 increase the apoptosis of A2780 and SKOV3. Therefore, OIN1 was shown to promote the growth of ovarian cancer cells through suppression of apoptosis.

＜＜ＯＩＮ１はアポトーシス関連遺伝子であるＲＡＳＳＦ５とＡＤＯＲＡ１の発現を制御する＞＞
　ＯＩＮ１がどのようにして卵巣がん細胞のアポトーシスを制御するのかに関して明らかにするために、卵巣がんにおけるＯＩＮ１の下流遺伝子の探索を行った。
　ＯＩＮ１の下流遺伝子を同定するために、まず高異型度漿液性卵巣がんの臨床検体（ｎ＝１５）においてＯＩＮ１の発現パターンと正又は負に相関した発現パターンを示す遺伝子を探索した。
　次に、見つかってきた遺伝子に富む生物学的パスウェイをＤＡＶＩＤ　Ｂｉｏｉｎｆｏｒｍａｔｉｃｓ　Ｒｅｓｏｕｒｃｅｓ　６．８により解析した。 << OIN1 regulates the expression of apoptosis-related genes RASSF5 and ADORA1 >>
To clarify how OIN1 regulates apoptosis of ovarian cancer cells, a search for downstream genes of OIN1 in ovarian cancer was performed.
In order to identify the downstream gene of OIN1, we first searched for a gene showing a positive or negative correlation with the expression pattern of OIN1 in a clinical sample (n = 15) of highly atypical serous ovarian cancer.
Next, the gene-rich biological pathways found were analyzed by DAVID Bioinformatics Resources 6.8.

　表１に、高異型度漿液性卵巣がんの検体において発現パターンがＯＩＮ１の発現パターンと正の相関関係を示す遺伝子とそれらの遺伝子に富んだ生物学的パスウェイをまとめたものを示し、表２に、高異型度漿液性卵巣がんの検体において発現パターンがＯＩＮ１の発現パターンと負の相関関係を示す遺伝子とそれらの遺伝子に富んだ生物学的パスウェイをまとめたものを示す。 Table 1 shows a summary of genes whose expression pattern positively correlates with the expression pattern of OIN1 in specimens of highly atypical serous ovarian cancer and biological pathways rich in those genes. Shown below is a summary of genes whose expression pattern negatively correlates with the expression pattern of OIN1 in a sample of highly atypical serous ovarian cancer and a biological pathway rich in those genes.

Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002

　表１に示したように、ＯＩＮ１の発現と正に相関する遺伝子は「Ｉｎｎｅｒ　ｃｅｌｌ　ｍａｓｓ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ」、「ＤＮＡ　ｒｅｐｌｉｃａｔｉｏｎ　ｉｎｉｔｉａｔｉｏｎ」、「Ｒｅｓｐｏｎｓｅ　ｔｏ　ＵＶ」、「Ｒｅｓｐｏｎｓｅ　ｔｏ　Ｘ－ｒａｙ」等の生物学的パスウェイに集積することが認められた。一方、表２に示したように、ＯＩＮ１の発現と負に相関する遺伝子は「Ｉｎｔｒａｃｅｌｌｕｌａｒ　ｓｉｇｎａｌ　ｔｒａｎｓｄｕｃｔｉｏｎ」、「Ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ａｐｏｐｔｏｔｉｃ　ｐｒｏｃｅｓｓ」、「Ｃｙｔｏｓｋｅｌｅｔｏｎ　ｏｒｇａｎｉｚａｔｉｏｎ」、「Ｎｅｇａｔｉｖｅ　ｒｅｇｕｌａｔｉｏｎ　ｏｆ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ」等の生物学的パスウェイへ集積した。 As shown in Table 1, the genes that positively correlate with the expression of OIN1 are biological such as "Inner cell mass cell proliferation", "DNA replication initiation", "Response to UV", and "Response to X-ray". It was allowed to accumulate in the pathway. On the other hand, as shown in Table 2, the genes that negatively correlate with the expression of OIN1 are "Intracellular signal transduction", "Regulation of apoptosis", "Cytoskeleton organization", "Negative cell biology", etc. Accumulated on the pathway.

－ＯＩＮ１の下流遺伝子の同定－
　表１及び２に記載した遺伝子から、細胞増殖やアポトーシスに関わるいくつかの遺伝子を選び出し、ＯＩＮ１のノックダウンがそれらの遺伝子の発現に与える影響に関して解析した。 -Identification of downstream gene of OIN1-
From the genes listed in Tables 1 and 2, several genes involved in cell proliferation and apoptosis were selected and analyzed for the effect of knockdown of OIN1 on the expression of those genes.

　ｓｉＯＩＮ１　＃１、ｓｉＯＩＮ１　＃２、ｓｉＣｏｎｔｒｏｌをトランスフェクションしたＡ２７８０において、各種遺伝子の相対的な発現量をｑＲＴ－ＰＣＲにより定量した結果（ｎ＝４）を図３Ａに示す。図３Ａ中、横軸は各種遺伝子を示し、遺伝子中の棒グラフは、それぞれ、ｓｉＣｏｎｔｒｏｌをトランスフェクションした場合（左）、ｓｉＯＩＮ１　＃１をトランスフェクションした場合（中央）、ｓｉＯＩＮ１　＃２をトランスフェクションした場合（右）の結果を示し、＊はｐ＜０．０５を表す。統計解析は２元配置分散分析により行った。 FIG. 3A shows the results (n = 4) of quantifying the relative expression levels of various genes by qRT-PCR in A2780 transfected with siOIN1 # 1, siOIN1 # 2, and siControl. In FIG. 3A, the horizontal axis shows various genes, and the bar graphs in the genes show the case of transfecting siControl (left), the case of transfecting siOIN1 # 1 (center), and the case of transfecting siOIN1 # 2, respectively. The result of (right) is shown, and * represents p <0.05. Statistical analysis was performed by two-way ANOVA.

　図３Ａに示したように、Ａ２７８０において、アポトーシスに関わる遺伝子であるＲＡＳＳＦ５とＡＤＯＲＡ１という遺伝子の発現が、ＯＩＮ１ノックダウンにより増加することが明らかとなった。 As shown in FIG. 3A, it was revealed that in A2780, the expression of genes involved in apoptosis, RASSF5 and ADORA1, was increased by OIN1 knockdown.

　また、ｓｉＯＩＮ１　＃１、ｓｉＯＩＮ１　＃２、ｓｉＣｏｎｔｒｏｌをトランスフェクションしたＳＫＯＶ３において、ＲＡＳＳＦ５遺伝子とＡＤＯＲＡ１遺伝子の相対的な発現量をｑＲＴ－ＰＣＲにより定量した結果を図３Ｂに示す（ｎ＝４）。図３Ｂ中、左側のグラフはＲＡＳＳＦ５遺伝子についての結果を示し、右側のグラフはＡＤＯＲＡ１遺伝子の結果を示す。図３Ｂ中、＊はｐ＜０．０５を表し、＊＊はｐ＜０．０１を表す。統計解析は２元配置分散分析により行った。 Further, the results of quantifying the relative expression levels of the RASSF5 gene and the ADORA1 gene by qRT-PCR in SKOV3 transfected with siOIN1 # 1, siOIN1 # 2, and siControl are shown in FIG. 3B (n = 4). In FIG. 3B, the graph on the left shows the results for the RASSF5 gene, and the graph on the right shows the results for the ADORA1 gene. In FIG. 3B, * represents p <0.05 and ** represents p <0.01. Statistical analysis was performed by two-way ANOVA.

　図３Ｂに示したようにＳＫＯＶ３おいてもＲＡＳＳＦ５遺伝子とＡＤＯＲＡ１遺伝子の発現がＯＩＮ１ノックダウンにより増加することが明らかとなった。 As shown in FIG. 3B, it was clarified that the expression of the RASSF5 gene and the ADORA1 gene was increased by OIN1 knockdown even in SKOV3.

　図３Ｃに、高異型度漿液性卵巣がんの臨床検体（ｎ＝１５）において、ＲＡＳＳＦ５遺伝子（上図）とＡＤＯＲＡ１遺伝子（下図）の発現パターンをＲＮＡシーケンシングのデータから解析した結果を示す。
　図３Ａ及び３Ｂの結果と一致するように、高異型度漿液性卵巣がんの臨床検体において、ＲＡＳＳＦ５とＡＤＯＲＡ１という遺伝子の発現パターンは、ＯＩＮ１の発現パターンと負に相関する傾向があることがＲＮＡシーケンシングのデータから示された。 FIG. 3C shows the results of analysis of the expression patterns of the RASSF5 gene (upper figure) and the ADORA1 gene (lower figure) from RNA-seqing data in a clinical specimen (n = 15) of highly atypical serous ovarian cancer.
As is consistent with the results in FIGS. 3A and 3B, RNA that the expression patterns of the genes RASSF5 and ADORA1 tend to be negatively correlated with the expression pattern of OIN1 in clinical specimens of highly atypical serous ovarian cancer. Shown from sequencing data.

　これらのことから、ＲＡＳＳＦ５とＡＤＯＲＡ１はＯＩＮ１の下流遺伝子であることが示唆された。 From these facts, it was suggested that RASSF5 and ADORA1 are downstream genes of OIN1.

　これまでの研究から、ＲＡＳＳＦ５は、ｍａｃｒｏｐｈａｇｅ　ｓｔｉｍｕｌａｔｉｎｇ　１／２（ＭＳＴ１／２）と結合し、ＭＳＴ１／２の活性化を引き起こし、細胞増殖やアポトーシスに影響を与えることが示唆されている（Ｔ．Ｊ．　Ｌｉａｏ，　Ｃ．Ｊ．　Ｔｓａｉ，　Ｈ．　Ｊａｎｇ，　Ｄ．　Ｆｕｓｈｍａｎ，　Ｒ．　Ｎｕｓｓｉｎｏｖ，　ＲＡＳＳＦ５：　Ａｎ　ＭＳＴ　ａｃｔｉｖａｔｏｒ　ａｎｄ　ｔｕｍｏｒ　ｓｕｐｐｒｅｓｓｏｒ　ｉｎ　ｖｉｖｏ　ｂｕｔ　ｏｐｐｏｓｉｔｅ　ｉｎ　ｖｉｔｒｏ，　Ｃｕｒｒ．　Ｏｐｉｎ．　Ｓｔｒｕｃｔ．　Ｂｉｏｌ．，　４１　（２０１６）　２１７－２２４，　ｄｏｉ：　１０．１０１６／ｊ．ｓｂｉ．２０１６．０９．００１．）。
　また、ＲＡＳＳＦ５は、ｔｕｍｏｒ　ｎｅｃｒｏｓｉｓ　ｆａｃｔｏｒ　α（ＴＮＦ－α）、ＴＮＦ－ｒｅｌａｔｅｄ　ａｐｏｐｔｏｓｉｓ－ｉｎｄｕｃｉｎｇ　ｌｉｇａｎｄ（ＴＲＡＩＬ）、ＣＤ４０　ｌｉｇａｎｄといった分子により誘導されるアポトーシスに関わることが報告されている（Ｊ．　Ｐａｒｋ，　Ｓ．Ｉ．　Ｋａｎｇ，　Ｓ．Ｙ．　Ｌｅｅ，　Ｘ．Ｆ．　Ｚｈａｎｇ，　Ｍ．Ｓ．　Ｋｉｍ，　Ｌ．Ｆ．　Ｂｅｅｒｓ，　Ｄ．Ｓ．　Ｌｉｍ，　Ｊ．　Ａｖｒｕｃｈ，　Ｈ．Ｓ．　Ｋｉｍ，　Ｓ．Ｂ．　Ｌｅｅ，　Ｔｕｍｏｒ　ｓｕｐｐｒｅｓｓｏｒ　ｒａｓ　ａｓｓｏｃｉａｔｉｏｎ　ｄｏｍａｉｎ　ｆａｍｉｌｙ　５　（ＲＡＳＳＦ５／ＮＯＲＥ１）　ｍｅｄｉａｔｅｓ　ｄｅａｔｈ　ｒｅｃｅｐｔｏｒ　ｌｉｇａｎｄ－ｉｎｄｕｃｅｄ　ａｐｏｐｔｏｓｉｓ，　Ｊ．　Ｂｉｏｌ．　Ｃｈｅｍ．，　２８５　（２０１０）　３５０２９－３５０３８，　ｄｏｉ：　１０．１０７４／ｊｂｃ．Ｍ１１０．１６５５０６．、Ｔ．　Ｅｌｍｅｔｗａｌｉ，　Ａ．　Ｓａｌｍａｎ，　Ｄ．Ｈ．　Ｐａｌｍｅｒ，　ＮＯＲＥ１Ａ　ｉｎｄｕｃｔｉｏｎ　ｂｙ　ｍｅｍｂｒａｎｅ－ｂｏｕｎｄ　ＣＤ４０Ｌ　（ｍＣＤ４０Ｌ）　ｃｏｎｔｒｉｂｕｔｅｓ　ｔｏ　ＣＤ４０Ｌ－ｉｎｄｕｃｅｄ　ｃｅｌｌ　ｄｅａｔｈ　ａｎｄ　Ｇ１　ｇｒｏｗｔｈ　ａｒｒｅｓｔ　ｉｎ　ｐ２１－ｍｅｄｉａｔｅｄ　ｍｅｃｈａｎｉｓｍ，　Ｃｅｌｌ　Ｄｅａｔｈ　Ｄｉｓ．，　７　（２０１６）　ｅ２１４６，　ｄｏｉ：　１０．１０３８／ｃｄｄｉｓ．２０１６．５２．）。
　重要なことに、ＲＡＳＳＦ５の発現増加によって卵巣がん細胞の増殖が抑制されることが示されている（Ｂ．Ｔ．　Ｌｉ，　Ｃ．　Ｙｕ，　Ｙ．　Ｘｕ，　Ｓ．Ｂ．　Ｌｉｕ，　Ｈ．Ｙ．　Ｆａｎ，　Ｗ．Ｗ．　Ｐａｎ，　ＴＥＴ１　ｉｎｈｉｂｉｔｓ　ｃｅｌｌ　ｐｒｏｌｉｆｅｒａｔｉｏｎ　ｂｙ　ｉｎｄｕｃｉｎｇ　ＲＡＳＳＦ５　ｅｘｐｒｅｓｓｉｏｎ，　Ｏｎｃｏｔａｒｇｅｔ，　８　（２０１７）　８６３９５－８６４０９，　ｄｏｉ：　１０．１８６３２／ｏｎｃｏｔａｒｇｅｔ．２１１８９．）。 Previous studies suggest that RASSF5 binds to macrophage structuring 1/2 (MST1 / 2), causes activation of MST1 / 2, and affects cell proliferation and apoptosis (TJ). Liao, CJ Tsai, H. Jang, D. Fushman, R. Nussinov, RASSF5: An MST activator and tumor suppressor in vivo apoptosis. -224, doi: 10.1016 / j.sbi. 2016.09.001.).
In addition, RASSF5 has been reported to be involved in molecule-induced apoptosis such as tumor necrosis factor α (TNF-α), TNF-related apoptosis-inducing led (TRAIL), and CD40 led (J. Park, S. I. Kang, S. Y. Lee, X. F. Zhang, MS Kim, LF Beers, DS Lim, J. Avruch, HS Kim, SB Lee, Tumor suppressor ras apoptosis domain family 5 (RASSF5 / NORE1) mediates death receptor lid-ind-indicated apoptosis, J. Biol. Chem. Elmetwalli, A. Salman, DH Palmer, NORE1A apoptosis by molecule-bound CD40L (mCD40L) conjunctions to CD40L-industred doi: 10.1038 / cddis. 2016.52.).
Importantly, increased expression of RASSF5 has been shown to suppress the proliferation of ovarian cancer cells (BT Li, C. Yu, Y. Xu, SB Liu, HY. Fan, WW Pan, TET1 inhibits cell proliferation by inducing RASSF5 expression, Oncotarget, 8 (2017) 86395-86409, doi: 10.18632 / oncottage.

　ＡＤＯＲＡ１は、７回膜貫通型のＧタンパク質共役受容体であり、細胞外領域のアデノシンの受容体である（Ｍ．Ｈ．　Ｋａｚｅｍｉ，　Ｓ．　Ｒａｏｏｆｉ　Ｍｏｈｓｅｎｉ，　Ｍ．　Ｈｏｊｊａｔ－Ｆａｒｓａｎｇｉ，　Ｅ．　Ａｎｖａｒｉ，　Ｇ．　Ｇｈａｌａｍｆａｒｓａ，　Ｈ．　Ｍｏｈａｍｍａｄｉ，　Ｆ．　Ｊａｄｉｄｉ－Ｎｉａｒａｇｈ，　Ａｄｅｎｏｓｉｎｅ　ａｎｄ　ａｄｅｎｏｓｉｎｅ　ｒｅｃｅｐｔｏｒｓ　ｉｎ　ｔｈｅ　ｉｍｍｕｎｏｐａｔｈｏｇｅｎｅｓｉｓ　ａｎｄ　ｔｒｅａｔｍｅｎｔ　ｏｆ　ｃａｎｃｅｒ，　Ｊ．　Ｃｅｌｌ．　Ｐｈｙｓｉｏｌ．，　２３３　（２０１８）　２０３２－２０５７，　ｄｏｉ：　１０．１００２／ｊｃｐ．２５８７３．）。
　がんにおけるＡＤＯＲＡ１の機能はがん種に応じて異なることが報告されている。卵巣がんにおいては、過去の研究からＡＤＯＲＡ１のアンタゴニストであるＳＬＶ３２０の処理がアデノシン処理条件下でのＡ２７８０の生存を促進する結果が示されており、これはＡＤＯＲＡ１が卵巣がんに対して抑制的な働きを持つ可能性を示唆している（Ｐ．　Ｓｕｒｅｅｃｈａｔｃｈａｉｙａｎ，　Ａ．　Ｈａｍａｃｈｅｒ，　Ｎ．　Ｂｒｏｃｋｍａｎｎ，　Ｂ．　Ｓｔｏｒｋ，　Ｍ．Ｕ．　Ｋａｓｓａｃｋ，　Ａｄｅｎｏｓｉｎｅ　ｅｎｈａｎｃｅｓ　ｃｉｓｐｌａｔｉｎ　ｓｅｎｓｉｔｉｖｉｔｙ　ｉｎ　ｈｕｍａｎ　ｏｖａｒｉａｎ　ｃａｎｃｅｒ　ｃｅｌｌｓ，　Ｐｕｒｉｎｅｒｇｉｃ　Ｓｉｇｎａｌ．，　１４　（２０１８）　３９５－４０８，　ｄｏｉ：　１０．１００７／ｓ１１３０２－０１８－９６２２－７．）。 ADORA1 is a 7-transmembrane G protein-coupled receptor and a receptor for adenosine in the extracellular space (MH Kazemi, S. Raofi Mohseni, M. Hojjat-Farsangi, E. Anvari, G. Ghamfarsa, H. Mohammadi, F. Jadidi-Niaargh, Adenosine and adenosine receptors in the immunopathogenesis in the imiunopathogenesis s. ).
It has been reported that the function of ADORA1 in cancer differs depending on the type of cancer. In ovarian cancer, previous studies have shown that treatment with SLV320, an antagonist of ADORA1, promotes the survival of A2780 under adenosine-treated conditions, which is that ADORA1 is inhibitory for ovarian cancer. (P. Sureechachiyan, A. Hamacher, N. Blockmann, B. Stalk, MU Kassack, Adenosine engine ovarian cypsy 2018) 395-408, doi: 10.1007 / s11302-018-9622-7.).

　以上のことから、ＯＩＮ１がＲＡＳＳＦ５やＡＤＯＲＡ１により誘導されるアポトーシスを抑制して卵巣がんの増殖を促進するというモデルが示唆された。 From the above, it was suggested that OIN1 suppresses apoptosis induced by RASSF5 and ADORA1 and promotes the growth of ovarian cancer.

＜＜ＯＩＮ１はｉｎ　ｖｉｖｏにおける卵巣腫瘍の増殖に重要な役割を果たす＞＞
　Ａ２７８０に由来する異種移植腫瘍モデルを用いてＯＩＮ１の病態生理学的意義を調べた。
　この解析のために、Ａ２７８０細胞をＭａｔｒｉｇｅｌと混合し、雌のヌードマウスに皮下注射した。その後、ｓｉＯＩＮ１　＃１を週に２回がんに注射してＡ２７８０由来異種移植腫瘍の増殖を１８日間観察した。
　結果を図４Ａ～４Ｆに示す。 << OIN1 plays an important role in the growth of ovarian tumors in vivo >>
The pathophysiological significance of OIN1 was investigated using a xenograft tumor model derived from A2780.
For this analysis, A2780 cells were mixed with Matrigel and injected subcutaneously into female nude mice. Then, siOIN1 # 1 was injected into the cancer twice a week and the growth of A2780-derived xenograft tumor was observed for 18 days.
The results are shown in FIGS. 4A-4F.

　図４Ａの上図は、ｓｉＣｏｎｔｒｏｌを注射した卵巣がん異種移植腫瘍モデルマウスの一例を示し、下図は、ｓｉＯＩＮ１　＃１を注射した卵巣がん異種移植腫瘍モデルマウスの一例を示す。 The upper figure of FIG. 4A shows an example of an ovarian cancer xenograft tumor model mouse injected with siControl, and the lower figure shows an example of an ovarian cancer xenograft tumor model mouse injected with siOIN1 # 1.

　図４Ｂに、各ｓｉＲＮＡを注射したＡ２７８０由来異種移植腫瘍の増殖を調べた結果を示す。図４Ｂでは、腫瘍の体積の平均値±ＳＤをグラフで示した（ｓｉＣｏｎｔｒｏｌ処理腫瘍はｎ＝７、ｓｉＯＩＮ１　＃１処理腫瘍はｎ＝８）。図４Ｂ中、＊＊はｐ＜０．０１、＊＊＊はｐ＜０．００１を表す。統計解析はＳｔｕｄｅｎｔのｔ検定により行った。なお、ｓｉＲＮＡは形成された異種移植腫瘍に週に２回注射した。 FIG. 4B shows the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA. In FIG. 4B, the mean value ± SD of the tumor volume is shown graphically (n = 7 for siControl-treated tumor, n = 8 for siOIN1 # 1 treated tumor). In FIG. 4B, ** represents p <0.01 and *** represents p <0.001. Statistical analysis was performed by Student's t-test. The siRNA was injected into the formed xenograft tumor twice a week.

　図４Ｃに、異種移植腫瘍を切除し、重量を測定した（ｓｉＣｏｎｔｒｏｌ処理腫瘍はｎ＝７、ｓｉＯＩＮ１　＃１処理腫瘍はｎ＝７）結果を示す。図４Ｃのグラフには腫瘍の重量の平均値±ＳＤを示した。図４Ｃ中、＊はｐ＜０．０５を表す。統計解析はＭａｎｎ－ＷｈｉｔｎｅｙのＵ検定により行った。 FIG. 4C shows the results of excision of xenograft tumor and weighing (n = 7 for siControl-treated tumor, n = 7 for siOIN1 # 1 treated tumor). The graph in FIG. 4C shows the mean ± SD of the tumor weight. In FIG. 4C, * represents p <0.05. Statistical analysis was performed by the Mann-Whitney U test.

　図４Ａ～４Ｃに示したように、ｓｉＯＩＮ１　＃１の注射はＡ２７８０由来異種移植腫瘍の体積と重量を有意に減少させることが示された。 As shown in FIGS. 4A-4C, injection of siOIN1 # 1 was shown to significantly reduce the volume and weight of A2780-derived xenograft tumors.

　また、腫瘍におけるＯＩＮ１、ＲＡＳＳＦ５、及びＡＤＯＲＡ１の発現を調べた結果を図４Ｄ～４Ｆに示す。 The results of examining the expression of OIN1, RASSF5, and ADORA1 in the tumor are shown in FIGS. 4D-4F.

　図４Ｄに、ｓｉＯＩＮ１　＃１注射によるＯＩＮ１のノックダウン効率をｑＲＴ－ＰＣＲにより解析した結果を示す。図４Ｄでは、ＯＩＮ１の相対的な発現量をＧＡＰＤＨ　ｍＲＮＡの発現量で補正して算出し、グラフにはｓｉＣｏｎｔｒｏｌ処理条件と比較したときのＯＩＮ１の発現量の倍率変化の平均値±ＳＤを示した（ｓｉＣｏｎｔｒｏｌ処理腫瘍はｎ＝３、ｓｉＯＩＮ１　＃１処理腫瘍はｎ＝３）。図４Ｄ中、＊はｐ＜０．０５を表す。統計解析はＳｔｕｄｅｎｔのｔ検定により行った。 FIG. 4D shows the results of analysis of the knockdown efficiency of OIN1 by injection of siOIN1 # 1 by qRT-PCR. In FIG. 4D, the relative expression level of OIN1 was corrected by the expression level of GAPDH mRNA and calculated, and the graph shows the average value ± SD of the change in the expression level of OIN1 when compared with the siControl treatment conditions. (N = 3 for siControl-treated tumors, n = 3 for siOIN1 # 1 treated tumors). In FIG. 4D, * represents p <0.05. Statistical analysis was performed by Student's t-test.

　図４Ｅ及び４Ｆに、Ａ２７８０由来異種移植腫瘍におけるＲＡＳＳＦ５（図４Ｅ）とＡＤＯＲＡ１（図４Ｆ）の相対的発現量をｑＲＴ－ＰＣＲにより解析した結果を示す。図４Ｄと同様に、各遺伝子の相対的な発現量をＧＡＰＤＨ　ｍＲＮＡの発現量で補正して算出し、グラフにはｓｉＣｏｎｔｒｏｌ処理条件と比較したときの各遺伝子の発現量の倍率変化の平均値±ＳＤを示した（ｓｉＣｏｎｔｒｏｌ処理腫瘍はｎ＝３、ｓｉＯＩＮ１　＃１処理腫瘍はｎ＝３）。図４Ｅ及び４Ｆ中、＊はｐ＜０．０５を表す。統計解析はＳｔｕｄｅｎｔのｔ検定により行った。 FIGS. 4E and 4F show the results of analysis of the relative expression levels of RASSF5 (FIG. 4E) and ADORA1 (FIG. 4F) in A2780-derived xenograft tumors by qRT-PCR. Similar to FIG. 4D, the relative expression level of each gene was corrected by the expression level of GAPDH mRNA and calculated. It showed SD (n = 3 for siControl-treated tumors, n = 3 for siOIN1 # 1 treated tumors). In FIGS. 4E and 4F, * represents p <0.05. Statistical analysis was performed by Student's t-test.

　図４Ｄ～４Ｆに示したように、ｓｉＯＩＮ１　＃１の注射により腫瘍におけるＯＩＮ１の発現が減少することを確認したが、その一方でＲＡＳＳＦ５やＡＤＯＲＡ１の発現は増加することを明らかにした。 As shown in FIGS. 4D-4F, it was confirmed that injection of siOIN1 # 1 decreased the expression of OIN1 in the tumor, but on the other hand, it was revealed that the expression of RASSF5 and ADORA1 was increased.

　以上の結果から、ＯＩＮ１は、ＲＡＳＳＦ５やＡＤＯＲＡ１の発現を制御してアポトーシスを抑制し、ｉｎ　ｖｉｖｏでの卵巣がんの増殖を促進することが示唆された。そのため、ＯＩＮ１を標的とする本発明の二本鎖核酸分子は、女性がんの核酸創薬として有用であることが示唆された。 From the above results, it was suggested that OIN1 regulates the expression of RASSF5 and ADORA1 to suppress apoptosis and promote the growth of ovarian cancer in vivo. Therefore, it was suggested that the double-stranded nucleic acid molecule of the present invention targeting OIN1 is useful as a nucleic acid drug discovery for female cancer.

＜＜ＯＩＮ１は子宮体がん及び乳がん細胞の増殖を促進する＞＞
　卵巣がん以外のがんにおけるＯＩＮ１の機能を探るために、子宮体がん細胞であるＩｓｈｉｋａｗａにおいてｓｉＲＮＡ（ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２）を使用したＯＩＮ１のノックダウン実験を行った。 << OIN1 promotes the growth of endometrial cancer and breast cancer cells >>
In order to investigate the function of OIN1 in cancers other than ovarian cancer, a knockdown experiment of OIN1 using siRNA (siOIN1 # 1 and siOIN1 # 2) was performed in Ishikawa, which is an endometrial cancer cell.

　ＩｓｈｉｋａｗａにｓｉＯＩＮ１　＃１又はｓｉＯＩＮ１　＃２を処理し、ＯＩＮ１のノックダウン効率をｑＲＴ－ＰＣＲにより解析した結果を図５Ａに示す。図５Ａでは、ＯＩＮ１の相対的な発現量をＧＡＰＤＨ　ｍＲＮＡの発現量で補正して算出し、グラフにはｓｉＣｏｎｔｒｏｌを処理した条件と比較したときのＯＩＮ１の発現倍率の変化の平均値±ＳＤを示した（ｎ＝３）。図５Ａ中、＊＊はｐ＜０．０１を表す。統計解析は２元配置分散分析により行った。 FIG. 5A shows the results of processing shiOIN1 # 1 or siOIN1 # 2 on Ishikawa and analyzing the knockdown efficiency of OIN1 by qRT-PCR. In FIG. 5A, the relative expression level of OIN1 is corrected by the expression level of GAPDH mRNA and calculated, and the graph shows the average value ± SD of the change in the expression ratio of OIN1 when compared with the condition treated with siControl. (N = 3). In FIG. 5A, ** represents p <0.01. Statistical analysis was performed by two-way ANOVA.

　図５Ａに示したように、ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２の処理が、ＩｓｈｉｋａｗａにおいてＯＩＮ１の発現を有意に減少させることが確認された。 As shown in FIG. 5A, it was confirmed that the treatment of siOIN1 # 1 and siOIN1 # 2 significantly reduced the expression of OIN1 in Ishikawa.

　また、ＯＩＮ１のノックダウンがＩｓｈｉｋａｗａの増殖を抑制することをＤＮＡアッセイにより調べた結果を図５Ｂに示す。図５ＢのグラフではＤＮＡ量の測定値の平均値±ＳＤを示した（ｎ＝５）。図５Ｂ中、＊＊＊はｐ＜０．０００１を表す。統計解析は２元配置分散分析により行った。 Further, FIG. 5B shows the results of examining by DNA assay that knockdown of OIN1 suppresses the proliferation of Ishikawa. In the graph of FIG. 5B, the average value ± SD of the measured values of the DNA amount is shown (n = 5). In FIG. 5B, *** represents p <0.0001. Statistical analysis was performed by two-way ANOVA.

　図５Ｂに示したように、ｓｉＯＩＮ１　＃１及びｓｉＯＩＮ１　＃２の処理が、Ｉｓｈｉｋａｗａの増殖を有意に抑制することが確認された。 As shown in FIG. 5B, it was confirmed that the treatment of siOIN1 # 1 and siOIN1 # 2 significantly suppressed the proliferation of Ishikawa.

　次に、乳がん細胞であるＢｒＣ－ＰＤＣにおいてｓｉＯＩＮ１　＃１を使用したＯＩＮ１のノックダウン実験を行った。 Next, a knockdown experiment of OIN1 using siOIN1 # 1 was performed on BrC-PDC, which is a breast cancer cell.

　ＢｒＣ－ＰＤＣにｓｉＯＩＮ１　＃１を処理し、ＯＩＮ１のノックダウン効率をｑＲＴ－ＰＣＲにより解析した結果を図５Ｃに示す。図５Ｃでは、ＯＩＮ１の相対的な発現量をＧＡＰＤＨ　ｍＲＮＡの発現量で補正して算出し、グラフにはｓｉＣｏｎｔｒｏｌを処理した条件と比較したときのＯＩＮ１の発現倍率の変化の平均値±ＳＤを示した（ｎ＝３）。図５Ｃ中、＊はｐ＜０．０５を表す。統計解析はＳｔｕｄｅｎｔのｔ検定により行った。 FIG. 5C shows the results of processing siOIN1 # 1 on BrC-PDC and analyzing the knockdown efficiency of OIN1 by qRT-PCR. In FIG. 5C, the relative expression level of OIN1 is corrected by the expression level of GAPDH mRNA, and the graph shows the average value ± SD of the change in the expression ratio of OIN1 when compared with the condition treated with siControl. (N = 3). In FIG. 5C, * represents p <0.05. Statistical analysis was performed by Student's t-test.

　図５Ｃに示したように、ｓｉＯＩＮ１　＃１の処理が、ＢｒＣ－ＰＤＣにおいてＯＩＮ１の発現を有意に減少させることが確認された。 As shown in FIG. 5C, it was confirmed that the treatment of siOIN1 # 1 significantly reduced the expression of OIN1 in BrC-PDC.

　また、ＯＩＮ１のノックダウンがＢｒＣ－ＰＤＣの増殖を抑制することをＣｅｌｌＴｉｔｅｒ－３Ｄ　Ｃｅｌｌ　Ｖｉａｂｉｌｉｔｙ　Ａｓｓａｙ（プロメガ社）により調べた結果を図５Ｄに示す。図５Ｄ中、＊＊はｐ＜０．００１を表す。統計解析はＳｔｕｄｅｎｔのｔ検定により行った。 Further, FIG. 5D shows the results of investigating that knockdown of OIN1 suppresses the proliferation of BrC-PDC by CellTiter-3D Cell Viability Assay (Promega). In FIG. 5D, ** represents p <0.001. Statistical analysis was performed by Student's t-test.

　図５Ｄに示したように、ｓｉＯＩＮ１　＃１の処理が、ＢｒＣ－ＰＤＣの増殖を有意に抑制することが確認された。 As shown in FIG. 5D, it was confirmed that the treatment of siOIN1 # 1 significantly suppressed the proliferation of BrC-PDC.

＜＜ＯＩＮ１の過剰発現は卵巣がん細胞の増殖を促進し、アポトーシス関連遺伝子の発現を抑制する＞＞
　次に卵巣がんにおけるＯＩＮ１の過剰発現による影響を解析した。 << Overexpression of OIN1 promotes the proliferation of ovarian cancer cells and suppresses the expression of apoptosis-related genes >>
Next, the effect of overexpression of OIN1 on ovarian cancer was analyzed.

　ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０及びＳＫＯＶ３細胞におけるＯＩＮ１　ＲＮＡの発現の変化を確認した結果を図６Ｃ（Ａ２７８０細胞）及び図６Ｆ（ＳＫＯＶ３細胞）に示す。データは平均±ＳＤ（ｎ＝３）として表した。図６Ｃ及び６Ｆ中、「Ｖｅｃｔｏｒ」は空のベクター、「ＯＩＮ１」はＯＩＮ１発現プラスミドでトランスフェクトした場合の結果を示す。また、図６Ｃ及び６Ｆ中の「＊＊＊」はｐ＜０．００１を示す（Ｓｔｕｄｅｎｔのｔ検定）。 The results of confirming changes in the expression of OIN1 RNA in A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector are shown in FIGS. 6C (A2780 cells) and 6F (SKOV3 cells). The data are expressed as mean ± SD (n = 3). In FIGS. 6C and 6F, "Vector" shows an empty vector, and "OIN1" shows the result when transfected with an OIN1 expression plasmid. Further, "***" in FIGS. 6C and 6F indicates p <0.001 (Student's t-test).

　図６Ｃ及び６Ｆに示したように、ＯＩＮ１発現プラスミドをＡ２７８０及びＳＫＯＶ３細胞にトランスフェクションすることにより、ＯＩＮ１の発現が有意に増加することがｑＲＴ－ＰＣＲにより確認された。 As shown in FIGS. 6C and 6F, it was confirmed by qRT-PCR that the expression of OIN1 was significantly increased by transfecting the OIN1 expression plasmid into A2780 and SKOV3 cells.

　ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０及びＳＫＯＶ３細胞の増殖を、ＤＮＡアッセイを用いて解析した結果を図６Ａ（Ａ２７８０細胞）及び図６Ｂ（ＳＫＯＶ３細胞）に示す。データは平均±ＳＤ（ｎ＝５）として表した。図６Ａ及び６Ｂ中、「Ｖｅｃｔｏｒ」は空のベクター、「ＯＩＮ１」はＯＩＮ１発現プラスミドでトランスフェクトした場合の結果を示す。また、図６Ａ及び６Ｂ中の「＊」はｐ＜０．０５、「＊＊＊」はｐ＜０．００１を示す（Ｓｔｕｄｅｎｔのｔ検定）。 The results of analysis of the proliferation of A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector using a DNA assay are shown in FIGS. 6A (A2780 cells) and 6B (SKOV3 cells). The data are expressed as mean ± SD (n = 5). In FIGS. 6A and 6B, "Vector" shows an empty vector, and "OIN1" shows the result when transfected with an OIN1 expression plasmid. Further, "*" in FIGS. 6A and 6B indicates p <0.05, and "***" indicates p <0.001 (Student's t-test).

　図６Ａ及び６Ｂに示したように、ＯＩＮ１の過剰発現は、卵巣がん細胞の増殖を有意に亢進させることが確認された。 As shown in FIGS. 6A and 6B, it was confirmed that overexpression of OIN1 significantly promoted the proliferation of ovarian cancer cells.

　ＯＩＮ１発現プラスミド又は空のベクターでトランスフェクトされたＡ２７８０及びＳＫＯＶ３細胞におけるＲＡＳＳＦ５　ｍＲＮＡ及びＡＤＯＲＡ１　ｍＲＮＡの発現の変化を確認した結果を図６Ｄ（Ａ２７８０細胞、ＲＡＳＳＦ５）、図６Ｅ（Ａ２７８０細胞、ＡＤＯＲＡ１）、図６Ｇ（ＳＫＯＶ３細胞、ＲＡＳＳＦ５）、及び図６Ｈ（ＳＫＯＶ３細胞、ＡＤＯＲＡ１）に示す。データは平均±ＳＤ（ｎ＝３）として表した。図６Ｄ、６Ｅ、６Ｇ、及び６Ｈ中、「Ｖｅｃｔｏｒ」は空のベクター、「ＯＩＮ１」はＯＩＮ１発現プラスミドでトランスフェクトした場合の結果を示す。また、図６Ｄ、６Ｅ、６Ｇ、及び６Ｈ中の「＊」はｐ＜０．０５、「＊＊」はｐ＜０．０１を示す（Ｓｔｕｄｅｎｔのｔ検定）。 The results of confirming changes in the expression of RASSF5 mRNA and ADORA1 mRNA in A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or an empty vector are shown in FIGS. 6D (A2780 cells, RASSF5), FIG. 6G (SKOV3 cells, RASSF5), and FIG. 6H (SKOV3 cells, ADORA1). The data are expressed as mean ± SD (n = 3). In FIGS. 6D, 6E, 6G, and 6H, "Vector" shows an empty vector, and "OIN1" shows the result when transfected with an OIN1 expression plasmid. Further, "*" in FIGS. 6D, 6E, 6G, and 6H indicates p <0.05, and "**" indicates p <0.01 (Student's t-test).

　図６Ｄ、６Ｅ、６Ｇ、及び６Ｈに示したように、ＯＩＮ１の過剰発現は、Ａ２７８０細胞とＳＫＯＶ３細胞においてＲＡＳＳＦ５とＡＤＯＲＡ１の発現を減少させることが確認された。 As shown in FIGS. 6D, 6E, 6G, and 6H, it was confirmed that overexpression of OIN1 reduced the expression of RASSF5 and ADORA1 in A2780 cells and SKOV3 cells.

＜＜ＯＩＮ１は子宮体がんで高発現する＞＞
　子宮体がんの３４検体に由来するＲＮＡシーケンシング解析より、ＯＩＮ１の発現量をＲＰＫＭ（ｒｅａｄｓ　ｐｅｒ　ｋｉｌｏｂａｓｅ　ｐｅｒ　ｍｉｌｌｉｏｎ　ｓｅｑｕｅｎｃｅｄ　ｒｅａｄｓ）の値として算出した。結果を図７に示す。 << OIN1 is highly expressed in endometrial cancer >>
The expression level of OIN1 was calculated as the value of RPKM (reads per kilobase per methylion complemented reads) from RNA sequencing analysis derived from 34 specimens of endometrial cancer. The results are shown in FIG.

　図７に示したように、卵巣がん検体と同等レベルにＯＩＮ１を高発現する子宮体がん検体が存在することが示された。 As shown in FIG. 7, it was shown that there is an endometrial cancer sample that highly expresses OIN1 at the same level as the ovarian cancer sample.

　以上の結果は、ＯＩＮ１が卵巣がんだけでなく、乳がんや、子宮がんの一例である子宮体がんといった女性がんの増殖にも重要であることを示しており、ＯＩＮ１が腫瘍マーカーや、女性がんの診断や治療の標的として有望であることが示された。
　また、本発明の二本鎖核酸分子により、卵巣がん細胞だけでなく、乳がん細胞や、子宮体がん細胞といった女性がん細胞の増殖も抑制することができることが示され、ＯＩＮ１を標的とする二本鎖核酸分子は、女性がんの核酸創薬に応用できる可能性が示唆された。 The above results indicate that OIN1 is important not only for ovarian cancer but also for the growth of female cancers such as breast cancer and endometrial cancer, which is an example of uterine cancer. , Has been shown to be a promising target for the diagnosis and treatment of female cancer.
It was also shown that the double-stranded nucleic acid molecule of the present invention can suppress the growth of not only ovarian cancer cells but also female cancer cells such as breast cancer cells and uterine body cancer cells, targeting OIN1. It was suggested that the double-stranded nucleic acid molecule could be applied to nucleic acid discovery of female cancer.

　本発明の態様としては、例えば、以下のものなどが挙げられる。
　＜１＞　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現を抑制するための二本鎖核酸分子であって、
（ａ）配列番号：２及び配列番号：３のいずれかで表される塩基配列からなる標的配列に対応する塩基配列を含むセンス鎖と、
（ｂ）前記（ａ）のセンス鎖と二本鎖を形成する該センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含むことを特徴とする二本鎖核酸分子である。
　＜２＞　二本鎖ＲＮＡ及び二本鎖ＲＮＡ－ＤＮＡキメラのいずれかである前記＜１＞に記載の二本鎖核酸分子である。
　＜３＞　ｓｉＲＮＡ及びキメラｓｉＲＮＡのいずれかである前記＜１＞から＜２＞のいずれかに記載の二本鎖核酸分子である。
　＜４＞　ｓｉＲＮＡである前記＜１＞から＜３＞のいずれかに記載の二本鎖核酸分子である。
　＜５＞　前記＜１＞から＜４＞のいずれかに記載の二本鎖核酸分子をコードする塩基配列を含むことを特徴とするＤＮＡである。
　＜６＞　前記＜５＞に記載のＤＮＡを含むことを特徴とするベクターである。
　＜７＞　前記＜１＞から＜４＞のいずれかに記載の二本鎖核酸分子、前記＜５＞に記載のＤＮＡ、及び前記＜６＞に記載のベクターの少なくともいずれかを含むことを特徴とする女性がん細胞増殖抑制剤である。
　＜８＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜７＞に記載の女性がん細胞増殖抑制剤である。
　＜９＞　女性がん細胞に、前記＜７＞から＜８＞のいずれかに記載の女性がん細胞増殖抑制剤を作用させることを特徴とする女性がん細胞の増殖抑制方法である。
　＜１０＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜９＞に記載の女性がん細胞の増殖抑制方法である。
　＜１１＞　前記＜１＞から＜４＞のいずれかに記載の二本鎖核酸分子、前記＜５＞に記載のＤＮＡ、及び前記＜６＞に記載のベクターの少なくともいずれかを含むことを特徴とする女性がんの腫瘍形成抑制剤である。
　＜１２＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜１１＞に記載の女性がんの腫瘍形成抑制剤である。
　＜１３＞　女性がんの腫瘍に、前記＜１１＞から＜１２＞のいずれかに記載の女性がんの腫瘍形成抑制剤を作用させることを特徴とする女性がんの腫瘍形成抑制方法である。
　＜１４＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜１３＞に記載の女性がんの腫瘍形成抑制方法である。
　＜１５＞　女性がんを予防乃至治療するための医薬であって、前記＜７＞から＜８＞のいずれかに記載の女性がん細胞増殖抑制剤及び前記＜１１＞から＜１２＞のいずれかに記載の女性がんの腫瘍形成抑制剤の少なくともいずれかを含むことを特徴とする医薬である。
　＜１６＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜１５＞に記載の医薬である。
　＜１７＞　個体に、前記＜１５＞から＜１６＞のいずれかに記載の医薬を投与することを特徴とする女性がんの予防乃至治療方法である。
　＜１８＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜１７＞に記載の女性がんの予防乃至治療方法である。
　＜１９＞　被検体由来の試料における配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの有無又はその発現量を指標として、前記被検体が女性がんに罹患しているか否か又は女性がんに罹患する可能性を有するか否かを評価することを含むことを特徴とする女性がんの評価方法である。
　＜２０＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜１９＞に記載の女性がんの評価方法である。
　＜２１＞　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを含むことを特徴とする女性がんの評価用マーカーである。
　＜２２＞　女性がんが、卵巣がん、子宮がん、及び乳がんの少なくともいずれかである前記＜２１＞に記載の女性がんの評価用マーカーである。 Examples of aspects of the present invention include the following.
<1> A double-stranded nucleic acid molecule for suppressing the expression of a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
(A) A sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3.
(B) A double-stranded nucleic acid molecule comprising the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand.
<2> The double-stranded nucleic acid molecule according to <1>, which is either a double-stranded RNA or a double-stranded RNA-DNA chimera.
<3> The double-stranded nucleic acid molecule according to any one of <1> to <2>, which is either siRNA or chimeric siRNA.
<4> The double-stranded nucleic acid molecule according to any one of <1> to <3>, which is siRNA.
<5> A DNA comprising a base sequence encoding the double-stranded nucleic acid molecule according to any one of <1> to <4>.
<6> A vector comprising the DNA according to <5>.
<7> It is characterized by containing at least one of the double-stranded nucleic acid molecule according to any one of <1> to <4>, the DNA according to the above <5>, and the vector according to the above <6>. It is a female cancer cell growth inhibitor.
<8> The female cancer cell growth inhibitor according to <7>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<9> A method for suppressing the growth of female cancer cells, which comprises allowing the female cancer cell to act on the female cancer cell growth inhibitor according to any one of <7> to <8>.
<10> The method for suppressing the growth of female cancer cells according to <9>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<11> It is characterized by containing at least one of the double-stranded nucleic acid molecule according to any one of <1> to <4>, the DNA according to the above <5>, and the vector according to the above <6>. It is a tumor formation inhibitor for female cancer.
<12> The tumor formation inhibitor for female cancer according to <11>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<13> A method for suppressing tumor formation of female cancer, which comprises allowing a tumor of female cancer to act on the tumor formation inhibitor of female cancer according to any one of <11> to <12>. ..
<14> The method for suppressing tumor formation of female cancer according to <13>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<15> A drug for preventing or treating female cancer, which is the female cancer cell growth inhibitor according to any one of <7> to <8> and any of the above <11> to <12>. It is a drug characterized by containing at least one of the tumor formation inhibitors for female cancers described in the above.
<16> The drug according to <15>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<17> A method for preventing or treating female cancer, which comprises administering the drug according to any one of <15> to <16> to an individual.
<18> The method for preventing or treating female cancer according to <17>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<19> Whether or not the subject suffers from female cancer using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index. Alternatively, it is a method for evaluating female cancer, which comprises evaluating whether or not the patient has a possibility of suffering from female cancer.
<20> The method for evaluating female cancer according to <19>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.
<21> A marker for evaluation of female cancer, which comprises a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
<22> The marker for evaluation of female cancer according to <21>, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.

Claims (8)

　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの発現を抑制するための二本鎖核酸分子であって、
（ａ）配列番号：２及び配列番号：３のいずれかで表される塩基配列からなる標的配列に対応する塩基配列を含むセンス鎖と、
（ｂ）前記（ａ）のセンス鎖と二本鎖を形成する該センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含むことを特徴とする二本鎖核酸分子。 It is a double-stranded nucleic acid molecule for suppressing the expression of a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
(A) A sense strand containing a base sequence corresponding to a target sequence consisting of a base sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3.
(B) A double-stranded nucleic acid molecule comprising the sense strand of (a) and an antisense strand containing a base sequence complementary to the sense strand forming the double strand. 　請求項１に記載の二本鎖核酸分子をコードする塩基配列を含むことを特徴とするＤＮＡ。 A DNA comprising a base sequence encoding the double-stranded nucleic acid molecule according to claim 1. 　請求項２に記載のＤＮＡを含むことを特徴とするベクター。 A vector comprising the DNA according to claim 2. 　請求項１に記載の二本鎖核酸分子、請求項２に記載のＤＮＡ、及び請求項３に記載のベクターの少なくともいずれかを含むことを特徴とする女性がん細胞増殖抑制剤。 A female cancer cell growth inhibitor comprising at least one of the double-stranded nucleic acid molecule according to claim 1, the DNA according to claim 2, and the vector according to claim 3. 　請求項１に記載の二本鎖核酸分子、請求項２に記載のＤＮＡ、及び請求項３に記載のベクターの少なくともいずれかを含むことを特徴とする女性がんの腫瘍形成抑制剤。 A tumor formation inhibitor for female cancer, which comprises at least one of the double-stranded nucleic acid molecule according to claim 1, the DNA according to claim 2, and the vector according to claim 3. 　女性がんを予防乃至治療するための医薬であって、請求項４に記載の女性がん細胞増殖抑制剤及び請求項５に記載の女性がんの腫瘍形成抑制剤の少なくともいずれかを含むことを特徴とする医薬。 A drug for preventing or treating female cancer, which comprises at least one of the female cancer cell growth inhibitor according to claim 4 and the tumor formation inhibitor for female cancer according to claim 5. A drug characterized by. 　被検体由来の試料における配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡの有無又はその発現量を指標として、前記被検体が女性がんに罹患しているか否か又は女性がんに罹患する可能性を有するか否かを評価することを含むことを特徴とする女性がんの評価方法。 Whether or not the subject has female cancer or whether or not the female has female cancer or not, using the presence or absence of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in the sample derived from the subject or the expression level thereof as an index. A method for assessing female cancer comprising assessing whether or not it has the potential to suffer from cancer. 　配列番号：１で表される塩基配列からなる長鎖非コードＲＮＡを含むことを特徴とする女性がんの評価用マーカー。 A marker for evaluating female cancer, which comprises a long non-coding RNA consisting of a base sequence represented by SEQ ID NO: 1.
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