Use of siRNA, DNA, vectors, inhibitors of female cancer cell proliferation, inhibitors of female cancer tumorigenesis, medicines, and long non-coding RNA

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

[Technical Field]

[0001] The present invention relates to a double-stranded nucleic acid molecule that can be suitably used for the prevention or treatment of female cancer, DNA containing a sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, an inhibitor of female cancer cell proliferation or an inhibitor of female cancer tumor formation containing at least one of the double-stranded nucleic acid molecule, the DNA, and the vector, a pharmaceutical containing at least one of the inhibitor of female cancer cell proliferation and the inhibitor of female cancer tumor formation, a method for evaluating female cancer using as an index the presence or absence or expression level of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1, and a marker for evaluating female cancer. [Background technology]

[0002] Ovarian cancer, a type of cancer affecting women, is one of the most common cancers affecting women (see Non-Patent Document 1). According to the GLOBOCAN 2018 database compiled by the International Agency for Research on Cancer (IARC), an external agency of the World Health Organization, ovarian cancer ranks eighth among all cancer types worldwide, with an estimated 295,414 new cases and 184,779 deaths (see Non-Patent Document 2). Despite ongoing advances in ovarian cancer treatment, ovarian cancer remains the most deadly cancer in women (see Non-Patent Document 3). While early-detected ovarian cancer treatments tend to be successful, early-stage ovarian cancer often exhibits few symptoms, and approximately 60% of cases are not diagnosed until the disease is more advanced. This is one of the reasons why the five-year survival rate for ovarian cancer patients is below 50% (see Non-Patent Documents 4-6). Therefore, addressing ovarian cancer is an urgent issue, and novel biomarkers and therapeutic targets are essential to improve ovarian cancer treatment outcomes.

[0003] With the recent advances in cDNA cloning and RNA sequencing technologies, it has become clear that in mammalian cells, including humans, approximately 70-90% of the genome is transcribed, resulting in various non-coding RNAs that do not code for proteins. Furthermore, it is gradually becoming clear that non-coding RNAs play important roles in various biological phenomena and diseases, including cancer.

[0004] Non-coding RNAs are classified by length, and non-coding RNAs longer than 200 bases are specifically defined as long non-coding RNAs (lncRNAs). The human genome is believed to contain a vast number of long non-coding RNA genes. For example, NONCODE (http: / / www.noncode.org / ), a database of non-coding RNAs expressed in 17 organisms, including humans and mice, lists as many as 96,308 human long non-coding RNA genes. While the functions of the majority of long non-coding RNAs remain unknown, some long non-coding RNAs have been shown to play important roles in the pathophysiology of cancer (see Non-Patent Documents 7-10). However, research into long non-coding RNAs is still in its infancy, and the functions of most long non-coding RNAs remain unknown. None of these RNAs have been clinically applied as markers or therapeutic targets in female cancers such as ovarian cancer. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] 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. [Non-patent document 2] CA Cancer J. Clin., 68 (2018) 394-424, doi: F. Bray, J. Ferlay, I. Soerjomataram, RL Siegel, LA Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries; 10.3322 / caac.21492.

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Non-licensed literature 9

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[0006] The present invention aims to solve the above-mentioned conventional problems and achieve the following objectives: Namely, the present invention aims to provide a double-stranded nucleic acid molecule that can effectively suppress the proliferation of female cancer cells and female cancer tumorigenesis and can be suitably used for the prevention or treatment of female cancer, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, an agent for inhibiting the proliferation of female cancer cells or an agent for inhibiting female cancer tumorigenesis that contains at least one of the double-stranded nucleic acid molecule, the DNA, and the vector, a medicine containing at least one of the agent for inhibiting the proliferation of female cancer cells and the agent for inhibiting female cancer tumorigenesis, a method for evaluating female cancer, and a marker for evaluating female cancer. [Means for solving the problem]

[0007] As a result of intensive research to achieve the above-mentioned object, the present inventors identified a long non-coding RNA (SEQ ID NO: 1, "ovarian long intergenic noncoding RNA 1" (hereinafter sometimes referred to as "OIN1") of unknown function that is overexpressed in female cancer specimens. Furthermore, they designed a double-stranded nucleic acid molecule that efficiently suppresses the expression of the long non-coding RNA, and found that introducing the double-stranded nucleic acid molecule into cancer cells or tumors suppresses the proliferation of the cancer cells and the formation of the tumors. Furthermore, they found that apoptosis is induced in cancer cells into which the double-stranded nucleic acid molecule has been introduced due to the decreased expression of the long non-coding RNA.

[0008] The means for solving the above problems are as follows: <1> A double-stranded nucleic acid molecule for inhibiting expression of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1, (a) a sense strand containing a nucleotide sequence corresponding to a target sequence consisting of a nucleotide 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 comprising a base sequence complementary to the sense strand, which forms a double strand. <2> The aforementioned <1> The DNA is characterized by comprising a base sequence encoding the double-stranded nucleic acid molecule described in <3> The aforementioned <2> A vector characterized by comprising the DNA described in <4> The aforementioned <1> The double-stranded nucleic acid molecule according to <2> and the DNA described in <3> and a female cancer cell proliferation inhibitor characterized by comprising at least one of the vectors described above. <5> In female cancer cells, <4> The present invention relates to a method for inhibiting the proliferation of female cancer cells, characterized by applying the female cancer cell proliferation inhibitor described in the above. <6> The aforementioned <1> The double-stranded nucleic acid molecule according to <2> and the DNA described in <3> and a tumor formation inhibitor for female cancer, characterized by comprising at least one of the vectors described above. <7> For female cancer tumors, <6> The present invention relates to a method for inhibiting tumor formation in female cancer, which comprises applying the tumor formation inhibitor for female cancer described in the above. <8> A pharmaceutical for preventing or treating female cancer, comprising: <4> The female cancer cell proliferation inhibitor described in <6> The present invention relates to a pharmaceutical composition comprising at least one of the tumor formation inhibitors for female cancers described above. <9> To the individual, <8> A method for preventing or treating female cancer, characterized by administering the pharmaceutical composition described in the above. <10> This is a method for evaluating female cancer, characterized by comprising evaluating whether a subject is suffering from female cancer or has the potential to be suffering from female cancer, using the presence or expression level of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample derived from the subject as an indicator. <11> This is a marker for evaluating female cancer, characterized by containing a long non-coding RNA consisting of the base sequence represented by SEQ ID NO:1. [Effects of the Invention]

[0009] The present invention can solve the above-mentioned problems of the prior art, and provides a double-stranded nucleic acid molecule that can effectively suppress the proliferation of female cancer cells and female cancer tumor formation and can be suitably used for the prevention or treatment of female cancer, DNA containing a base sequence encoding the double-stranded nucleic acid molecule, a vector containing the DNA, a female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor containing at least one of the double-stranded nucleic acid molecule, the DNA, and the vector, a pharmaceutical containing at least one of the female cancer cell proliferation inhibitor and the female cancer tumor formation inhibitor, a method for evaluating female cancer, and a marker for evaluating female cancer. [Brief explanation of the drawings]

[0010] [Figure 1A] FIG. 1A shows the results of comparing the expression levels of OIN1 in normal ovarian tissues, ovarian clear cell carcinoma specimens, and high-grade serous ovarian cancer specimens. [Figure 1B] Figure 1B shows the mapping of RNA sequencing reads from normal ovarian tissues and specimens from ovarian clear cell carcinoma and high-grade serous ovarian cancer to the OIN1 locus. [Figure 1C] FIG. 1C shows the results of examining OIN1 expression in ovarian cancer cells by qRT-PCR. [Figure 2A]FIG. 2A shows the results of qRT-PCR analysis of the OIN1 knockdown efficiency by siRNA against OIN1 in A2780 (left), SKOV3 (center), and RMG1 (right). [Figure 2B] FIG. 2B shows the results of a DNA assay demonstrating that knockdown of OIN1 suppresses proliferation of A2780 (left), SKOV3 (center), and RMG1 (right). [Figure 2C] FIG. 2C is a diagram showing the results of apoptosis analysis using A2780 cells. [Figure 2D] FIG. 2D is a diagram showing the results of apoptosis analysis using SKOV3 cells. [Figure 2E] FIG. 2E is a diagram showing the results of apoptosis analysis using A2780 cells. [Figure 2F] FIG. 2F is a diagram showing the results of apoptosis analysis using SKOV3 cells. [Figure 3A] Figure 3A shows the results of an analysis of the effect of OIN1 knockdown on the expression of various genes. [Figure 3B] Figure 3B is Figure 2 showing the results of an analysis of the effect of OIN1 knockdown on the expression of various genes. [Figure 3C] Figure 3C shows the results of analyzing the expression patterns of the RASSF5 gene (upper panel) and the ADORA1 gene (lower panel) from RNA sequencing data in clinical specimens of high-grade serous ovarian cancer. [Figure 4A] FIG. 4A shows an example of an ovarian cancer xenograft tumor model mouse injected with each siRNA. [Figure 4B] FIG. 4B shows the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA. [Figure 4C] FIG. 4C shows the results of excising and measuring the weight of xenograft tumors. [Figure 4D] FIG. 4D shows the results of examining OIN1 expression in A2780-derived xenograft tumors injected with each siRNA. [Figure 4E] FIG. 4E shows the results of examining the expression of RASSF5 in A2780-derived xenograft tumors injected with each siRNA. [Figure 4F] FIG. 4F shows the results of examining the expression of ADORA1 in A2780-derived xenograft tumors injected with each siRNA. [Figure 5A] FIG. 5A shows the results of treating Ishikawa with siOIN1 #1 or siOIN1 #2 and analyzing the OIN1 knockdown efficiency by qRT-PCR. [Figure 5B] FIG. 5B shows the results of a DNA assay demonstrating that knockdown of OIN1 suppresses the proliferation of Ishikawa. [Figure 5C] Figure 5C shows the results of treating BrC-PDC with siOIN1 #1 and analyzing the OIN1 knockdown efficiency by qRT-PCR. [Figure 5D] FIG. 5D shows the results of an investigation using CellTiter-3D Cell Viability Assay (Promega) showing that knockdown of OIN1 suppresses the proliferation of BrC-PDC. [Figure 6A] FIG. 6A shows the results of analyzing the proliferation of A2780 cells transfected with an OIN1 expression plasmid or an empty vector using a DNA assay. [Figure 6B] FIG. 6B shows the results of analyzing the proliferation of SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector using a DNA assay. [Figure 6C] FIG. 6C shows the results of confirming changes in OIN1 RNA expression in A2780 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 6D] FIG. 6D shows the results of confirming changes in RASSF5 mRNA expression in A2780 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 6E]FIG. 6E shows the results of confirming changes in ADORA1 mRNA expression in A2780 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 6F] FIG. 6F shows the results of confirming changes in OIN1 RNA expression in SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 6G] FIG. 6G shows the results of examining changes in RASSF5 mRNA expression in SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 6H] FIG. 6H shows the results of examining changes in ADORA1 mRNA expression in SKOV3 cells transfected with an OIN1 expression plasmid or an empty vector. [Figure 7] FIG. 7 shows the results of comparing the expression levels of OIN1 in normal ovarian tissues and endometrial cancer specimens. DETAILED DESCRIPTION OF THE INVENTION

[0011] (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 non-coding RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1, and comprises: (a) a sense strand comprising a nucleotide sequence corresponding to a target sequence consisting of the nucleotide sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3; and (b) an antisense strand comprising a nucleotide sequence complementary to the sense strand, which forms a duplex with the sense strand of (a). In the present invention, the term "double-stranded nucleic acid molecule" refers to a double-stranded nucleic acid molecule in which a sense strand and an antisense strand are hybridized.

[0012] <Long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1> The long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 was registered as NONHSAT013448 in the NONCODE database (NONCODE. Available online: http: / / www.noncode.org / (accessed on 31 May 2020).), but no research had been conducted on it until now, and its function was unknown. Therefore, the present inventors named the long non-coding RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 ovarian cancer long intergenic non-coding RNA 1 (OIN1). As shown in the test examples described below, OIN1 is overexpressed in female cancer cells and has the function of suppressing apoptosis of female cancer cells and promoting proliferation of female cancer cells.

[0013] 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. Therefore, in this specification, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is sometimes referred to as the "target RNA" of the double-stranded nucleic acid molecule.

[0014] <Sense strand, antisense strand> As a result of extensive investigations, the present inventors have found that a double-stranded nucleic acid molecule comprising an antisense strand containing a nucleotide sequence complementary to a specific target sequence (a nucleotide sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3) among the sequences of long non-coding RNAs consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a significantly superior expression-inhibitory effect on long non-coding RNAs consisting of the nucleotide sequence represented by SEQ ID NO: 1. Therefore, the double-stranded nucleic acid molecule of the present invention comprises (a) a sense strand containing a nucleotide sequence corresponding to a target sequence consisting of the nucleotide sequence represented by either SEQ ID NO: 2 or SEQ ID NO: 3, and (b) an antisense strand containing a nucleotide sequence complementary to the sense strand, which forms a duplex with the sense strand of (a). Here, the sense strand and the antisense strand may be RNA strands or RNA-DNA chimeric strands, and the sense strand and the antisense strand can hybridize with each other to form the double-stranded nucleic acid molecule.

[0015] The sense strand in the double-stranded nucleic acid molecule may contain a base sequence corresponding to the target sequence, or may contain other base sequences, or may consist only of a base sequence corresponding to the target sequence. Furthermore, the antisense strand in the double-stranded nucleic acid molecule may contain a base sequence complementary to the sense strand to the extent that it can hybridize with the sense strand, and may also contain other base sequences, but it preferably contains 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more of a base sequence complementary to the sense strand.

[0016] <Type> The type of the double-stranded nucleic acid molecule is not particularly limited and can be appropriately selected depending on the purpose. Examples include double-stranded RNA (dsRNA) and double-stranded RNA-DNA chimera. Here, "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 "double-stranded RNA-DNA chimera" refers to a double-stranded nucleic acid molecule in which both the sense strand and the antisense strand are composed of chimeric sequences of RNA and DNA.

[0017] The double-stranded RNA and double-stranded RNA-DNA chimera are preferably siRNA (small interfering RNA) or chimeric siRNA, and more preferably siRNA.

[0018] Here, siRNA is a small double-stranded RNA of 18 to 29 bases in length, which has the function of cleaving target RNA having a sequence complementary to the antisense strand (guide strand) of the siRNA, thereby suppressing the expression of the target RNA. The siRNA is not particularly limited in its terminal structure, and can be appropriately selected depending on the purpose, as long as it has the sense strand and antisense strand as described above and can suppress the expression of the target RNA. For example, the siRNA may have a blunt end or a protruding end (overhang). In particular, the siRNA preferably has a structure in which the 3'-end of each strand overhangs by 2 to 6 bases, and more preferably has a structure in which the 3'-end of each strand overhangs by 2 bases.

[0019] Chimeric siRNA refers to a small double-stranded RNA-DNA chimera of 18 to 29 bases in length, in which a portion of the RNA sequence of the siRNA has been converted to DNA. Among these, a small double-stranded RNA-DNA chimera of 21 to 23 bases in length, in which within 8 bases on the 3' side of the sense strand of the siRNA and within 6 bases on the 5' side of the antisense strand have been converted to DNA, is preferred. The chimeric siRNA has the function of suppressing target gene expression, similar to the siRNA. The chimeric siRNA also includes a form in which a portion of the sequence converted to DNA has been converted back to RNA. As with the siRNA, the terminal structure of the chimeric siRNA is not particularly limited and can be appropriately selected depending on the purpose; for example, it may have a blunt end or a protruding end (overhang).

[0020] Specific examples of the siRNA include the following.

[0021] An example of an siRNA in which the target sequence is the base sequence represented by SEQ ID NO: 2 is an siRNA consisting of a sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 5 below. Sense strand 5'-GCUCAGCUCACGGCUUCUACC-3' (SEQ ID NO: 4) Antisense strand 5'-UAGAAGCCGUGAGCUGAGCUC-3' (SEQ ID NO: 5)

[0022] Furthermore, an example of an siRNA in which the target sequence is the base sequence represented by SEQ ID NO: 3 is an siRNA consisting of a sense strand of SEQ ID NO: 6 and an antisense strand of SEQ ID NO: 7 below. Sense strand 5'-GACAGGAGACUCCAGAAAAGG-3' (SEQ ID NO: 6) Antisense strand 5'-UUUUCUGGAGUCUCCUGUCUG-3' (SEQ ID NO: 7)

[0023] The double-stranded RNA may also be shRNA (short hairpin RNA). Here, 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. When expressed in vivo, shRNA forms base pairs to become a hairpin-shaped double-stranded RNA. The shRNA is then cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to inhibit the expression of target RNA. The terminal structure of the shRNA, like the siRNA and double-stranded RNA-DNA chimera, is not particularly limited and can be selected appropriately depending on the purpose; for example, it may have a blunt end or a protruding end (overhang).

[0024] <qualification> The double-stranded nucleic acid molecule may be appropriately modified depending on the purpose. For example, to confer resistance to nucleic acid degrading enzymes (nucleases) and improve stability in culture media or in vivo, the double-stranded nucleic acid molecule may be modified with 2'O-methylation, phosphorothioate (S-modification), LNA (Locked Nucleic Acid), or the like. Furthermore, to increase the efficiency of introduction into cells, the 5' or 3' end of the sense strand of the double-stranded nucleic acid molecule may be modified with nanoparticles, cholesterol, cell membrane-penetrating peptides, or the like. The method for modifying the double-stranded nucleic acid molecule in this manner is not particularly limited, and conventionally known techniques can be used as appropriate.

[0025] <How to obtain> The method for obtaining the double-stranded nucleic acid molecule is not particularly limited, and the molecule can be prepared based on a conventionally known method. For example, the siRNA can be prepared by chemically synthesizing 18- to 29-base-long single-stranded RNAs corresponding to the desired sense and antisense strands using an existing automated DNA / RNA synthesizer or the like, and then annealing them. Pre-annealed double-stranded siRNAs can be commercially available, or can be obtained by requesting synthesis from an siRNA synthesis contract company. Alternatively, a desired siRNA expression vector, such as the vector of the present invention described below, can be constructed, and the expression vector can be introduced into cells, allowing intracellular reactions to be utilized to produce siRNA. Furthermore, the chimeric siRNA can be prepared, for example, by chemically synthesizing the sense strand and antisense strand of a chimeric nucleic acid molecule and then annealing them.

[0026] (DNA, vector) The DNA of the present invention is a DNA containing a base sequence encoding the above-described double-stranded nucleic acid molecule of the present invention, and the vector of the present invention is a vector containing the DNA.

[0027] <dna> The DNA is not particularly limited as long as it contains a base sequence encoding the double-stranded nucleic acid molecule of the present invention and can be selected appropriately depending on the purpose, but it is preferable that a promoter sequence for controlling the transcription of the double-stranded nucleic acid molecule is linked upstream (5' side) of the base sequence encoding the double-stranded nucleic acid molecule. The promoter sequence is not particularly limited and can be selected appropriately depending on the purpose, and examples include pol II promoters such as the CMV promoter, and pol III promoters such as the H1 promoter and U6 promoter. Furthermore, it is more preferable that a terminator sequence for terminating 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 can be appropriately selected depending on the 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 of the DNA. The transcription unit can be constructed using a conventionally known method.

[0028] <Vector> The vector is not particularly limited as long as it contains the DNA and can be appropriately selected depending on the purpose, and examples thereof include a plasmid vector, a viral vector, etc. 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 can be appropriately selected depending on the purpose. For example, methods for expressing siRNA as a double-stranded nucleic acid molecule include a method of expressing two short single-stranded RNAs (tandem type) and a method of expressing a single-stranded RNA as shRNA (hairpin type). The tandem siRNA expression vector contains a DNA sequence encoding the sense strand and a DNA sequence encoding the antisense strand that constitute the siRNA, and contains DNA in which a promoter sequence is linked upstream (5' side) of the DNA sequence encoding each strand, and a terminator sequence is linked downstream (3' side) of the DNA sequence encoding each strand. Furthermore, the hairpin siRNA expression vector contains DNA in which the DNA sequence encoding the sense strand and the DNA sequence encoding the antisense strand that constitute the siRNA are arranged in opposite directions, the sense strand DNA sequence and the antisense strand DNA sequence are connected via a loop sequence, and a promoter sequence is linked upstream (5' side) and a terminator sequence is linked downstream (3' side) of them. Each of the vectors can be constructed using a conventionally known method, for example, by ligating the DNA to the cleavage site of a vector that has been previously cleaved with a restriction enzyme.

[0029] By introducing (transfecting) the DNA or the vector into a cell, the promoter is activated, and the double-stranded nucleic acid molecule can be generated. For example, in the tandem vector, the DNA is transcribed in the cell to generate a sense strand and an antisense strand, which hybridize to generate siRNA. In the hairpin vector, the DNA is transcribed in the cell to first generate a hairpin RNA (shRNA), which is then processed by Dicer to generate siRNA.

[0030] (An inhibitor of female cancer cell proliferation, an inhibitor of female cancer tumor formation) The female cancer cell proliferation inhibitor of the present invention is intended to inhibit the proliferation 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 contains other components as necessary. The tumor formation inhibitor for female cancer of the present invention is intended to inhibit tumor formation 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 further contains other components as necessary.

[0031] <Double-stranded nucleic acid molecules, DNA, vectors> Details of the double-stranded nucleic acid molecule are as described above in the section on the double-stranded nucleic acid molecule of the present invention. Because the double-stranded nucleic acid molecule can effectively inhibit the expression of the target long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1, it is suitable as an active ingredient of the female cancer cell proliferation inhibitor for inhibiting the proliferation of female cancer cells, or as an active ingredient of the female cancer tumorigenesis inhibitor for inhibiting the formation of female cancer tumors. Furthermore, details of the DNA and vector are as described above in the section on the DNA and 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 proliferation inhibitor or female cancer tumorigenesis inhibitor is not particularly limited and can be appropriately selected depending on the purpose. Furthermore, the female cancer cell proliferation inhibitor or female cancer tumorigenesis inhibitor may be at least one of the double-stranded nucleic acid molecule, DNA, and vector itself.

[0032] <Other ingredients> The other components are not particularly limited and can be appropriately selected depending on the purpose. Examples include diluents such as physiological saline or culture medium for diluting at least one of the double-stranded nucleic acid molecule, DNA, and vector to a desired concentration, and transfection reagents for introducing (transfecting) at least one of the double-stranded nucleic acid molecule, DNA, and vector into target cells or tumors. The content of the other ingredients in the female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor is not particularly limited and can be appropriately selected depending on the purpose.

[0033] <Women's cancer> The female cancers to which the female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor is applied are not particularly limited and can be selected appropriately depending on the purpose, but preferred examples include at least one of ovarian cancer, uterine cancer (including endometrial cancer and cervical cancer), and breast cancer. The types of ovarian cancer, uterine cancer, and breast cancer are not particularly limited and can be appropriately selected depending on the purpose.

[0034] The female cancer cells may be cells cultured in vitro or cells present in the body of an individual.

[0035] <effect> The female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor can be introduced into female cancer cells or female cancer tumors, for example, to allow the inhibitor to act on the female cancer cells or female cancer tumors. The method of introduction is not particularly limited and can be appropriately selected from conventionally known methods depending on the purpose, and examples include a method using a transfection reagent, a method using electroporation, a method using magnetic particles, a method utilizing viral infection, and a method of injection. The amount of the female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor to be applied to the female cancer cells or female cancer tumors is not particularly limited and can be appropriately selected depending on the type of cells or tumors, the degree of the desired effect, etc., but is, for example, 1 × 10 6 The amount of the active ingredient (the double-stranded nucleic acid molecule) per cell is preferably about 0.1 μg, more preferably about 5 μg, and particularly preferably about 15 μg.

[0036] The female cancer cell proliferation inhibitor or female cancer tumor formation inhibitor of the present invention may be used alone, or the two may be used in combination, or may be used in combination with other female cancer therapeutic agents.

[0037] (Method for inhibiting the proliferation of female cancer cells, method for inhibiting tumor formation in female cancer) The female cancer cell proliferation inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, and when allowed to act on female cancer cells, it can effectively inhibit the proliferation of female cancer cells through the suppression of expression of the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. Therefore, the present invention also relates to a method for inhibiting the proliferation of female cancer cells, which comprises allowing the female cancer cell proliferation inhibitor to act on female cancer cells. Furthermore, since the agent for inhibiting tumor formation in female cancer contains at least one of the double-stranded nucleic acid molecule, DNA, and vector, by acting on a female cancer tumor, it can effectively inhibit tumor formation in female cancer through suppression of expression of the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. Therefore, the present invention also relates to a method for inhibiting tumor formation in female cancer, which comprises acting the agent for inhibiting tumor formation in female cancer on a female cancer tumor. The female cancer is not particularly limited, and examples thereof include those described in the section <Female cancer> above (Female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor). In addition, in the method for inhibiting the proliferation of female cancer cells or the method for inhibiting tumor formation in female cancer, other therapeutic drugs for female cancer may be further administered.

[0038] (Pharmaceuticals) The pharmaceutical of the present invention is a pharmaceutical for preventing or treating female cancer, and contains at least one of the female cancer cell proliferation inhibitor and female cancer tumor formation inhibitor of the present invention described above, and further contains other ingredients as necessary. In the present invention, the term "prevention" refers to preventing the onset or recurrence of female cancer, and the term "treatment" refers to curing or alleviating the symptoms of cancer or preventing its progression. The female cancer is not particularly limited, and examples thereof include those described in the section <Female cancer> above (Female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor).

[0039] <Inhibitor of female cancer cell proliferation, inhibitor of female cancer tumor formation> Details of the female cancer cell proliferation inhibitor and female cancer tumor formation inhibitor are as described above in the section of the present invention (female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor).

[0040] The female cancer cell proliferation inhibitor contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, and can effectively inhibit the proliferation of female cancer cells through suppression of the expression of the targeted long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. That is, the female cancer cell proliferation inhibitor can be suitably used as a medicine for preventing or treating female cancer.

[0041] The tumor formation inhibitor for female cancers contains at least one of the double-stranded nucleic acid molecule, DNA, and vector of the present invention described above, and can effectively suppress tumor formation in female cancers through suppression of the expression of the targeted long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1. That is, the tumor formation inhibitor for female cancer can be suitably used as a medicine for preventing or treating female cancer.

[0042] The total content of at least one of the female cancer cell proliferation inhibitor and the female cancer tumor formation inhibitor in the pharmaceutical is not particularly limited and can be appropriately selected depending on the purpose. In addition, the pharmaceutical may consist of at least one of the female cancer cell proliferation inhibitor and the female cancer tumor formation inhibitor.

[0043] Here, the double-stranded nucleic acid molecule serving as the active ingredient of the pharmaceutical may be an unmodified double-stranded nucleic acid molecule itself, but it is preferable to use a double-stranded nucleic acid molecule in a form suitable for administration to a living body so that an appropriate preventive or therapeutic effect can be obtained. For example, the double-stranded nucleic acid molecule is preferably modified to enhance the stability of the double-stranded nucleic acid molecule in vivo. The types of modifications that can be made to the double-stranded nucleic acid molecule are not particularly limited, and examples include 2'O-methylation, phosphorothioate (S-modification), and LNA (Locked Nucleic Acid) modification. Furthermore, for the purpose of increasing the efficiency of introduction into target cells, it is also preferable to modify the 5' or 3' end of the sense strand of the double-stranded nucleic acid molecule with nanoparticles, cholesterol, cell membrane-penetrating peptides, etc. The method for making the double-stranded nucleic acid molecule modified is not particularly limited, and conventionally known techniques can be used as appropriate. In addition, in order to increase the efficiency of introduction of the double-stranded nucleic acid molecule into target cells, it is also preferable that the double-stranded nucleic acid molecule forms a complex with a liposome, a polymer matrix, etc. The method for forming the complex is not particularly limited, and any conventionally known method can be used as appropriate.

[0044] <Other ingredients> The other components are not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include pharmaceutically acceptable carriers. The carrier is also not particularly limited and can be selected appropriately depending on, for example, the dosage form. Furthermore, the content of the other components in the medicament is also not particularly limited and can be selected appropriately depending on the purpose.

[0045] <Dosage form> The dosage form of the pharmaceutical is not particularly limited and can be appropriately selected depending on, for example, the desired administration method. Examples include oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations to be dissolved when used, etc.), ointments, patches, gels, creams, external powders, sprays, and inhalation powders.

[0046] The oral solid preparation can be produced by a conventional method, for example, by adding an excipient to the active ingredient, and further adding additives such as a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, and an odorant, as needed. Examples of the excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Examples of the disintegrants include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose. Examples of the lubricants include purified talc, stearates, borax, and polyethylene glycol. Examples of the colorants include titanium oxide and iron oxide. Examples of the flavorings and fragrances include sucrose, orange peel, citric acid, and tartaric acid.

[0047] The oral liquid preparation can be produced by adding additives such as flavoring agents, buffering agents, and stabilizers to the active ingredient in a conventional manner. Examples of the flavoring and fragrance correcting agents include sucrose, orange peel, citric acid, tartaric acid, etc. Examples of the buffering agents include sodium citrate, etc. Examples of the stabilizers include tragacanth, gum arabic, gelatin, etc.

[0048] As the injection, for example, a pH adjusting agent, a buffering agent, a stabilizer, an isotonic agent, a local anesthetic, etc. can be added to the active ingredient, and an injection for subcutaneous, intramuscular, intravenous, etc. can be produced by a conventional method. Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, and sodium phosphate. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

[0049] The ointment can be prepared, for example, by combining the active ingredient with a known base, stabilizer, humectant, preservative, etc., and mixing them in a conventional manner. Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin, etc. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.

[0050] The patch can be produced, for example, by applying the ointment in the form of a cream, gel, paste, or the like to a known support in a conventional manner. Examples of the support include woven fabrics made of cotton, staple fiber, and chemical fibers, nonwoven fabrics, films made of soft vinyl chloride, polyethylene, polyurethane, and the like, and foam sheets.

[0051] <Administration> The medicament is suitable for preventing or treating female cancers. Therefore, the medicament can be preferably used by administering it to individuals who have been affected with or may have been affected with female cancers.

[0052] The individual to which the pharmaceutical is to be administered is not particularly limited and can be appropriately selected depending on the purpose. Examples include humans, mice, rats, cows, pigs, monkeys, dogs, and cats, with humans being particularly preferred.

[0053] The method of administering the pharmaceutical is not particularly limited, and either local administration or systemic administration can be selected depending on, for example, the dosage form of the pharmaceutical, the type of disease, the condition of the patient, etc. For example, in local administration, the active ingredient of the pharmaceutical (the double-stranded nucleic acid molecule) can be administered by directly injecting it into a desired site (e.g., a tumor site). For the injection, a conventionally known method such as injection can be used as appropriate. Furthermore, in systemic administration (e.g., oral administration, intraperitoneal administration, administration into the blood, etc.), it is preferable to appropriately apply a conventionally known drug delivery technique so that the active ingredient of the pharmaceutical (the double-stranded nucleic acid molecule) is delivered stably and efficiently to a desired site (e.g., a tumor site).

[0054] The dosage of the pharmaceutical is not particularly limited and can be appropriately selected depending on the age, body weight, desired degree of effect, etc. of the patient to be administered; for example, the amount of the active ingredient (the double-stranded nucleic acid molecule) per day administered to an adult is preferably 1 mg to 100 mg. Furthermore, there is no particular limitation on the number of times the drug is administered, and it can be appropriately selected depending on the age, body weight, desired degree of effect, etc. of the patient to whom the drug is administered.

[0055] The timing of administration of the medicament is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, administered prophylactically or therapeutically against a disease. In particular, since the medicament has an excellent effect of inhibiting the proliferation of female cancer cells and suppressing the formation of tumors in female cancer, it is considered desirable to administer the medicament at as early a stage as possible of the disease.

[0056] The medicament of the present invention may be used in combination with other therapeutic agents for female cancer.

[0057] (Methods for preventing or treating female cancers) The medicament contains at least one of the female cancer cell proliferation inhibitor and the female cancer tumor formation inhibitor, and when administered to an individual who has or may have had female cancer, it can effectively inhibit at least one of the proliferation of female cancer cells and the formation of female cancer tumors through the suppression of expression of the targeted long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1, thereby preventing or treating female cancer. Therefore, the present invention also relates to a method for preventing or treating female cancer, which comprises administering the medicament to an individual. The female cancer is not particularly limited, and examples thereof include those described in the section <Female cancer> above (Female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor). In addition, in the above-mentioned prevention or treatment method, other therapeutic drugs for female cancer may be further administered.

[0058] (Methods for assessing female cancers) The method for evaluating female cancer of the present invention includes at least an evaluation step, and may include other steps such as a detection step as necessary. The female cancer is not particularly limited, and examples thereof include those described in the section <Female cancer> above (Female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor).

[0059] <Evaluation process> The evaluation step is a step of evaluating whether or not the subject is suffering from female cancer or has the potential to suffer from female cancer, using the presence or absence or expression level of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample derived from the subject as an indicator.

[0060] -Sample derived from subject- The sample derived from the subject is not particularly limited as long as it is prepared from the subject individual and can be appropriately selected depending on the purpose, and examples thereof include cells, tissues, and blood from a lesion site. The sample may further be subjected to treatment such as RNA preparation. Only one type of sample may be used, or two or more types may be used.

[0061] The subject is not particularly limited and can be appropriately selected depending on the purpose. Examples include humans, mice, rats, cows, pigs, monkeys, dogs, and cats, with humans being particularly preferred.

[0062] -Long non-coding RNA consisting of the 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 as described in the section <Long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1> in the above-mentioned (double-stranded nucleic acid molecule) of the present invention.

[0063] -evaluation- If a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is detected in the sample, if the expression level of the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is higher than the expression level in a sample derived from a healthy individual, or if the expression level of the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is equal to or higher than the expression level in a sample derived from an individual suffering from female cancer, the individual is assessed as having or at risk of suffering from female cancer.

[0064] <Detection process> The detection step is a step of detecting a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample prepared from an individual.

[0065] -detection- The detection method is not particularly limited, and a known method can be appropriately selected depending on the purpose, and examples thereof include a method using qRT-PCR, a method using RNA sequencing, etc. These methods may be used alone or in combination of two or more.

[0066] The primer set used in 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, and can be selected appropriately depending on the purpose.

[0067] An example of the primer set is the following primer set. Forward 5'-TCTTCACCCCTAACCAGCAGGAA-3' (SEQ ID NO: 12) Reverse 5'-AGGACTGAAGTAAGTCCTGATGC-3' (SEQ ID NO: 13)

[0068] <Other processes> The other steps are not particularly limited and can be appropriately selected depending on the purpose. For example, the other steps include a step of preparing a sample to be used in the detection step.

[0069] 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 in diagnosis, and a method for predicting the onset of female cancer.

[0070] (marker for assessing female cancer) The marker for evaluating female cancer of the present invention contains at least a long non-coding RNA consisting of the base sequence shown in SEQ ID NO: 1, and may contain other components as necessary.

[0071] As shown in the test examples described below, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is overexpressed in female cancer cells. Furthermore, the long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 is involved in promoting the proliferation and suppressing apoptosis of female cancer cells through regulating the expression of the RASSF5 gene and the ADORA1 gene. Therefore, a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample collected from an individual can be used as a marker for evaluating female cancer. The sample is not particularly limited and can be appropriately selected depending on the purpose.

[0072] The female cancer is not particularly limited, and examples thereof include those described in the section <Female cancer> above (Female cancer cell proliferation inhibitor, female cancer tumor formation inhibitor).

[0073] The other components are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose. The marker for evaluating female cancer may include other markers for evaluating female cancer. [Example]

[0074] Test examples of the present invention will be explained below, but the present invention is not limited to these test examples.

[0075] (Test Example 1) Materials and Methods <<RNA sequencing analysis of clinical specimens from normal ovarian tissue, ovarian tumors, and endometrial cancer>> Patient clinical information and RNA sequencing experiments using clinical specimens were approved by the Saitama Medical University International Medical Center IRB (#13-165). High-grade serous ovarian carcinoma (HGSOC) specimens (n ​​= 15), ovarian clear cell carcinoma (OCCC) specimens (n ​​= 6), normal tissue specimens (n ​​= 6), or endometrial cancer specimens (n ​​= 34) were obtained from patients who underwent surgery for primary ovarian tumors with informed consent (#12-096).

[0076] RNA was extracted from the tissues immediately after freezing using NucleoSpin RNA (Takara). The quality of the RNA was analyzed using a bioanalyzer (Agilent). The RIN value of all RNA was 7 or higher. An RNA library was prepared using a SureSelect Strand Specific RNA Library Preparation Kit (Agilent), and RNA sequencing was performed using a HiSeq2500 (registered trademark) (Illumina) under the condition of 100 bp paired end.

[0077] The resulting FASTQ sequence files were aligned to the reference human genome (hg19) (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.).

[0078] Genes positively or negatively correlated with OIN1 expression in high-grade serous ovarian cancer samples were determined using correlation coefficients (r) and p-values. Next, we defined genes with positive and negative correlations based on the correlation coefficient (0.6 ≤ r ≤ 1 or -1 ≤ r ≤ -0.45, respectively). Biological pathways significantly containing these genes were analyzed using the database for annotation, visualization and integrated discovery (DAVID) Bioinformatics Resources 6.8 (https: / / david.ncifcrf.gov / summary.jsp).

[0079] <<Cell culture>> The human ovarian cancer cells used were A2780, ES2, OV90, OVCAR3, RMG1, and SKOV3. As uterine cancer cells, Ishikawa, which are human uterine cancer cells, were used. As breast cancer cells, BrC-PDC, which are human breast cancer cells established from clinical specimens of breast cancer patients, were used.

[0080] OV90, OVCAR3, SKOV3, and Ishikawa were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin under the conditions of 5% CO2 and 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 under the conditions of 5% CO2 and 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% CO2 and 37 °C. BrC-PDC was cultured in StemPro hESC SFM medium (manufactured by Thermo Fisher Scientific) containing 8.8 ng / mL basic fibroblast growth factor (manufactured by Thermo Fisher Scientific), 20 μmol / L Y-27632 (Rho-associated coiled-coil forming kinase inhibitor, manufactured by Thermo Fisher Scientific), 50 U / mL penicillin, and 50 μg / mL streptomycin in an Ultra-Low Attachment Multiple Well Plate (manufactured by Corning) under the conditions of 5% CO2 and 37 °C.

[0081] <<Transfection of siRNA and plasmid>> As double-stranded nucleic acid molecules against OIN1, small interference RNA (siRNA) was designed, 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 sequences of each siRNA are shown below.

[0082] [siOIN1 #1] -Target sequence- 5'-GCTCAGCTCACGGCTTCTACC-3' (SEQ ID NO: 2) -Sequence of double-stranded nucleic acid molecule (siRNA)- Sense strand 5'-GCUCAGCUCACGGCUUCUACC-3' (SEQ ID NO: 4) Antisense strand 5'-UAGAAGCCGUGAGCUGAGCUC-3' (SEQ ID NO: 5)

[0083] [siOIN1 #2] -Target sequence- 5'-GACAGGAGACTCCAGAAAAGG-3' (SEQ ID NO: 3) -Sequence of double-stranded nucleic acid molecule (siRNA)- Sense strand 5'-GACAGGAGACUCCAGAAAAGG-3' (SEQ ID NO: 6) Antisense strand 5'-UUUUCUGGAGUCUCCUGUCUG-3' (SEQ ID NO: 7)

[0084] [siControl] -Sequence of double-stranded nucleic acid molecule (siRNA)- Sense strand 5'-GUACCGCACGUCAUUCGUAUC-3' (SEQ ID NO: 8) Antisense strand 5'-GAUACGAAUGACGUGCGGUAC-3' (SEQ ID NO: 9)

[0085] Before transfection with the siRNA or the plasmid expressing the OIN1 gene, 3 × 10 cells were transfected in the case of A2780 and RMG1. 5 For SKOV3 cells, 1 x 10 5 Cells were seeded one by one into the wells of a 6-well plate. After culturing for 24 hours, the siRNA was transfected into the cells using Lipofectamine RNAiMax (manufactured by Thermo Fisher Scientific) so that the final concentration was 10 nM. Transfection of the plasmid expressing the OIN1 gene or the empty vector was performed using FuGene HD Transfection Reagent (manufactured by Promega). The transfection method followed the protocol of the product used. The cells were harvested 48 or 72 hours after transfection and used for the quantitative RT-PCR experiment described below.

[0086] <<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.). Tumor disruption was performed using Polytron PT3100 (manufactured by Kinematica). Single-stranded cDNA was synthesized by reverse transcription from 1 μg of extracted RNA using SuperScript III (manufactured by Thermo Fisher Scientific) and random hexamer primers. qRT-PCR was performed using the prepared cDNA, KAPA SYBR FAST qPCR Kit (manufactured by KAPA Biosystems), and a set of primers specific to the gene on a StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific). The expression level of RNA was analyzed by the ΔΔCt method according to the protocol of the StepOnePlus Real-Time PCR System product, and the expression level of the GAPDH gene was used for correction. The primers used for qRT-PCR are as follows. <​​​​​5'-GGTGGTCTCCTCTGACTTCAACA-3' (SEQ ID NO: 10) Reverse 5'-GTGGTCGTTGAGGGCAATG-3' (SEQ ID NO: 11) [OIN1] Forward 5'-TCTTCACCCCTAACCAGCAGGAA-3' (SEQ ID NO: 12) Reverse 5'-AGGACTGAAGTAAGTCCTGATGC-3' (SEQ ID NO: 13) [MCM5] Forward 5'-AGCATTCGTAGCCTGAAGTCG-3' (SEQ ID NO: 14) Reverse 5'-CGGCACTGGATAGAGATGCG-3' (SEQ ID NO: 15) [E2F3] Forward 5'-AGAAAGCGGTCATCAGTACCT-3' (SEQ ID NO: 16) Reverse 5'-TGGACTTCGTAGTGCAGCTCT-3' (SEQ ID NO: 17) [PIK3CB] Forward 5'-CTGCCTGCGACAGATGAGTG-3' (SEQ ID NO: 18) Reverse 5'-TCCGATTACCAAGTGCTCTTTC-3' (SEQ ID NO: 19) [NOTCH1] Forward 5'-GAGGCGTGGCAGACTATGC-3' (SEQ ID NO: 20) Reverse 5'-CTTGTACTCCGTCAGCGTGA-3' (SEQ ID NO: 21) [CDKN1B] Forward 5'-TAATTGGGGCTCCGGCTAACT-3' (SEQ ID NO: 22) Reverse 5'-TGCAGGTCGCTTCCTTATTCC-3' (SEQ ID NO: 23) [RASSF5] Forward 5'-GGGCATGAAACTGAGTGAAGA-3' (SEQ ID NO: 24) Reverse 5'-TGGCATCATAGATGGACTGGG-3' (SEQ ID NO: 25) [ADORA1] Forward 5'-CCACAGACCTACTTCCACACC-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)

[0088] <<Analysis of cell proliferation>> The cells were seeded in wells of a 96-well plate at 3,000 cells each for A2780 and RMG1, 1,000 cells each for SKOV3, and 2,000 cells each for Ishikawa. After 24 hours of culture, the cells were transfected with siRNA at a final concentration of 10 nM using Lipofectamine RNAiMax (Thermo Fisher Scientific). The cells were harvested 1, 3, and 5 days after seeding. Transfection of the OIN1 gene-expressing plasmid or empty vector was performed using FuGene HD Transfection Reagent (Promega). The cells were harvested 1 and 3 days after seeding. To assess cell proliferation, DNA in 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 using a 2030 ARVO X5 Multilabel Plate Reader (PerkinElmer) (DNA assay).

[0089] For BrC-PDCs, 40,000 cells were suspended in 240 μL of Opti-MEM medium (Thermo Fisher Scientific) and seeded into a 24-well plate (Ultra-Low Attachment Multiple Well Plate, Corning). Immediately afterwards, the cells were transfected with siRNA to a final concentration of 10 nM using Lipofectamine RNAiMax (Thermo Fisher Scientific). Six hours after transfection, 200 μL of StemPro hESC SFM medium (Thermo Fisher Scientific) was added. Five days after seeding, the cells were harvested, and the cells from each well were then equally divided and seeded into five wells of a 96-well plate. Then, to evaluate cell proliferation, the ATP content of the cells in the wells was measured using CellTiter-Glo 3D Cell Viability Assay (Promega) and TriStar 2 Measurement was carried out using an S LB 942 Multimode Reader (manufactured by Berthold Technologies).

[0090] <<Analysis of apoptosis using annexin V and propidium iodide (PI)>> In the case of A2780, 3×10 5 cells per well, and in the case of SKOV3, 1×10 5 cells per well were seeded in the wells of a 6-well plate. After culturing for 24 hours, siRNA was transfected into the cells using Lipofectamine RNAiMax so that the final concentration was 10 nM, and the cells were collected 72 hours later. Cells that had undergone apoptosis were stained using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to the product protocol. Cells stained with annexin V and PI were analyzed using a BD FACSCalibur (BD Biosciences).

[0091] <<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 CLEA Japan, Inc. A2780 (1×10 5 cells) and an equal volume of Matrigel matrix (Corning) were mixed and subcutaneously injected into the flanks of 10-week-old female nude mice. Thereafter, the mice were randomly divided into two groups. siControl or siOIN1 #1 (5 μg each) was mixed with the transfection reagent GeneSilencer reagent (Gene Therapy System) and injected into the tumors formed in the mice twice a week. The tumor volume was measured once a week and calculated using the formula 0.5×(diameter of axis 1)×(diameter of axis 2)×(diameter of axis 3).

[0092] <<Statistical analysis>> Statistical analysis of data was performed using the Mann-Whitney U test, two-way analysis of variance (two-way ANOVA), or Student's t test. The test method used is indicated for each data item. Microsoft Excel (Microsoft) and JMP 9.0.00 (SAS Institute) were used for statistical analysis.

[0093] <Result> Ovarian cancer long intergenic noncoding RNA 1 (OIN1) is highly expressed in ovarian cancer cells To explore long non-coding RNAs specifically expressed in ovarian cancer, we performed RNA sequencing analysis using normal ovarian tissue (Normal, n=6) and clinical specimens from ovarian clear cell carcinoma (OCCC, n=6) and high-grade serous ovarian cancer (HGSOC, n=15). As a result, we found that a long non-coding RNA registered as NONHSAT013448 in the NONCODE database (NONCODE. Available online: http: / / www.noncode.org / (accessed on 31 May 2020).) was highly expressed in ovarian clear cell carcinoma and high-grade serous ovarian carcinoma compared to normal ovarian tissue (see Figure 1A and B).

[0094] Figure 1A shows the comparison of the expression levels of the long non-coding RNA (OIN1) registered as NONHSAT013448 in ovarian normal tissue, ovarian clear cell carcinoma, and high-grade serous ovarian carcinoma. The expression levels of the long non-coding RNA registered as NONHSAT013448 were higher in ovarian clear cell carcinoma and high-grade serous ovarian carcinoma compared to normal tissue. The expression level of the long non-coding RNA registered as NONHSAT013448 was calculated using RPKM (reads per kilobase per million sequenced reads) (JY Wang, AQ Lu, LJ Chen, LncRNAs in ovarian cancer, Clin. Chim. Acta, 490 (2019) 17-27, doi: 10.1016 / j.cca.2018.12.013). In Figure 1A, * indicates p<0.05, and ** indicates p<0.01. Statistical analysis was performed using the Mann-Whitney U test.

[0095] The long non-coding RNA, registered as NONHSAT013448, is transcribed from the gene registered as NONHSAG005930 in the NONCODE database. The NONHSAG005930 gene is located on the long arm of human chromosome 10 (10q21.1), and is separated from the nearest protein-coding genes, protocadherin-related 15 (PCDH15) and mannose binding lectin 2 (MBL2), by approximately 0.78 Mb and 0.20 Mb, respectively. Therefore, since the NONHSAG005930 gene does not overlap with any protein-coding genes, NONHSAT013448 is classified as a long intergenic non-coding RNA.

[0096] The function of NONHSAT013448 has not been studied and is unclear so far. Therefore, this long non-coding RNA was named ovarian cancer long intergenic noncoding RNA 1 (OIN1). The nucleotide sequence of OIN1 was as shown in SEQ ID NO: 1.

[0097] The upper figure in Fig. 1B is a diagram mapping the reads of RNA sequencing derived from normal ovarian tissue and specimens of ovarian clear cell carcinoma and high-grade serous ovarian carcinoma to the locus of OIN1, and the lower figure is a diagram showing that OIN1 is composed of two exons.

[0098] Next, the expression of OIN1 in ovarian cancer cells was examined by qRT-PCR.

[0099] Fig. 1C is a diagram showing the result of analyzing the expression level of OIN1 in ovarian cancer cells by qRT-PCR and correcting it with the expression level of GAPDH mRNA. In Fig. 1C, the average value ± SD of the expression level of OIN1 is shown (n = 3).

[0100] As shown in Fig. 1C, it was revealed that OIN1 was highly expressed in A2780, SKOV3, and RMG1, and weakly expressed in OV90 cells (see Fig. 1C). Since OIN1 was highly expressed, A2780, SKOV3, and RMG1 were used for the functional analysis experiment of OIN1.

[0101] <<OIN1 promotes the proliferation of ovarian cancer cells and inhibits 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 conducted.

[0102] Figure 2A shows the results of qRT-PCR analysis of OIN1 knockdown efficiency using siRNAs against OIN1 (siOIN1 #1 and siOIN1 #2) in A2780 (left), SKOV3 (center), and RMG1 (right). The relative expression levels of OIN1 were calculated by correcting for GAPDH mRNA expression levels. Figure 2A shows the mean ± SD of the fold change in OIN1 expression compared to siControl (n = 3). *** indicates p < 0.0001. Statistical analysis was performed using two-way ANOVA.

[0103] As shown in Figure 2A, treatment with siOIN1 #1 and siOIN1 #2 significantly reduced OIN1 expression in A2780, SKOV3, and RMG1.

[0104] Furthermore, DNA assay confirmed that knockdown of OIN1 by siOIN1 #1 and siOIN1 #2 significantly suppressed the proliferation of these ovarian cancer cells. The results are shown in Figure 2B.

[0105] In Figure 2B, the left shows the results for A2780, the center shows the results for SKOV3, and the right shows the results for RMG1. Figure 2B shows the mean ± SD of the measured DNA content (n = 5 for A2780, n = 3 for SKOV3, and n = 3 for RMG1). In Figure 2B, ** indicates p < 0.001 and *** indicates p < 0.0001. Statistical analysis was performed using two-way analysis of variance.

[0106] These results indicate that OIN1 plays an important role in ovarian cancer proliferation.

[0107] Next, to clarify how OIN1 regulates ovarian cancer proliferation, we investigated the effect of OIN1 knockdown on apoptosis in ovarian cancer cells.

[0108] The results of apoptosis analysis using a technique combining cell staining with PI and Annexin V and flow cytometry are shown in Figures 2C to 2F.

[0109] The results when using A2780 as the cell are shown in Figures 2C and 2E, and the results when using SKOV3 as the cell are shown in Figures 2D and 2F. As shown in Figures 2C and 2D, it was revealed by cell staining with PI and annexin V and flow cytometry that the knockdown of OIN1 promotes apoptosis of A2780 and SKOV3. Also, in Figures 2E and 2F, the proportion of cells that underwent apoptosis in A2780 and SKOV3 was quantified and shown in a graph. In Figures 2E and 2F, ** represents p < 0.001, and *** represents p < 0.0001. Statistical analysis was performed by two-way analysis of variance.

[0110] As described above, siOIN1 #1 and siOIN1 #2 were shown to increase apoptosis of A2780 and SKOV3. Therefore, it was shown that OIN1 promotes the growth of ovarian cancer cells through the suppression of apoptosis.

[0111] <<OIN1 controls the expression of RASSF5 and ADORA1, which are apoptosis-related genes>> In order to clarify how OIN1 controls apoptosis of ovarian cancer cells, a search for downstream genes of OIN1 in ovarian cancer was conducted. To identify the downstream genes of OIN1, first, genes showing an expression pattern positively or negatively correlated with the expression pattern of OIN1 were searched in clinical specimens (n = 15) of high-grade serous ovarian cancer. Next, the biological pathways rich in the found genes were analyzed by DAVID Bioinformatics Resources 6.⑧.

[0112] Table 1 shows a summary of genes whose expression patterns show a positive correlation with the expression pattern of OIN1 and the biological pathways rich in those genes in specimens of high-grade serous ovarian cancer, and Table 2 shows a summary of genes whose expression patterns show a negative correlation with the expression pattern of OIN1 and the biological pathways rich in those genes in specimens of high-grade serous ovarian cancer.

[0113] [Table 1]

[0114] [Table 2]

[0115] As shown in Table 1, genes positively correlated with OIN1 expression were found to be clustered in biological pathways such as "Inner cell mass cell proliferation," "DNA replication initiation," "Response to UV," and "Response to X-ray." On the other hand, as shown in Table 2, genes negatively correlated with OIN1 expression were clustered in biological pathways such as "Intracellular signal transduction," "Regulation of apoptotic process," "Cytoskeleton organization," and "Negative regulation of cell proliferation."

[0116] -Identification of downstream genes of OIN1- From the genes listed in Tables 1 and 2, several genes involved in cell proliferation and apoptosis were selected and the effect of OIN1 knockdown on the expression of these genes was analyzed.

[0117] Figure 3A shows the results (n = 4) of quantification of the relative expression levels of various genes by qRT-PCR in A2780 cells transfected with siOIN1 #1, siOIN1 #2, and siControl. In Figure 3A, the horizontal axis indicates the various genes, and the bar graphs within the genes show the results for transfection with siControl (left), siOIN1 #1 (center), and siOIN1 #2 (right), respectively. * indicates p < 0.05. Statistical analysis was performed using two-way ANOVA.

[0118] As shown in Figure 3A, in A2780, the expression of RASSF5 and ADORA1 genes, which are involved in apoptosis, was found to be increased by OIN1 knockdown.

[0119] Figure 3B shows the results of qRT-PCR quantification of the relative expression levels of the RASSF5 gene and ADORA1 gene in SKOV3 cells transfected with siOIN1 #1, siOIN1 #2, and siControl (n = 4). In Figure 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 Figure 3B, * indicates p < 0.05, and ** indicates p < 0.01. Statistical analysis was performed using two-way ANOVA.

[0120] As shown in Figure 3B, OIN1 knockdown also increased the expression of RASSF5 and ADORA1 genes in SKOV3.

[0121] Figure 3C shows the results of RNA sequencing analysis of the expression patterns of the RASSF5 gene (top panel) and ADORA1 gene (bottom panel) in clinical specimens of high-grade serous ovarian cancer (n = 15). Consistent with the results in Figures 3A and 3B, RNA sequencing data showed that the expression patterns of the genes RASSF5 and ADORA1 tended to negatively correlate with the expression pattern of OIN1 in clinical specimens of high-grade serous ovarian cancer.

[0122] These findings suggest that RASSF5 and ADORA1 are downstream genes of OIN1.

[0123] Previous studies have suggested that RASSF5 binds to macrophage stimulating 1 / 2 (MST1 / 2), activating MST1 / 2 and affecting cell proliferation and apoptosis (TJ Liao, CJ 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.). RASSF5 has also been reported to be involved in apoptosis induced by molecules such as tumor necrosis factor α (TNF-α), TNF-related apoptosis-inducing ligand (TRAIL), and CD40 ligand (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.). Importantly, increased expression of RASSF5 has been shown to suppress ovarian cancer cell proliferation (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 / oncotarget.21189.).

[0124] ADORA1 is a seven-transmembrane G protein-coupled receptor and an extracellular adenosine receptor (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.). The function of ADORA1 in cancer has been reported to vary depending on the cancer type. In ovarian cancer, previous studies have shown that treatment with the ADORA1 antagonist SLV320 promotes survival of A2780 cells under adenosine treatment, suggesting that ADORA1 may have an inhibitory role in ovarian cancer (P. Sureechatchaiyan, A. Hamacher, N. Brockmann, B. Stork, MU Kassack, Adenosine enhances cisplatin sensitivity in human ovarian cancer cells, Purinergic Signal., 14 (2018) 395-408, doi: 10.1007 / s11302-018-9622-7.).

[0125] From the above, a model was suggested that OIN1 suppresses apoptosis induced by RASSF5 and ADORA1 and promotes the growth of ovarian cancer.

[0126] <<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 subcutaneously injected into female nude mice. Then, siOIN1 #1 was injected into the cancer twice a week, and the growth of A2780-derived xenograft tumors was observed for 18 days. The results are shown in FIGS. 4A to 4F.

[0127] The upper figure in FIG. 4A shows an example of a mouse with an ovarian cancer xenograft tumor model injected with siControl, and the lower figure shows an example of a mouse with an ovarian cancer xenograft tumor model injected with siOIN1 #1.

[0128] FIG. 4B shows the results of examining the growth of A2780-derived xenograft tumors injected with each siRNA. In FIG. 4B, the average value ± SD of the tumor volume is shown in a graph (n = 7 for siControl-treated tumors, n = 8 for siOIN1 #1-treated tumors). 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 tumors twice a week.

[0129] FIG. 4C shows the results of excising the xenograft tumors and measuring their weights (n = 7 for siControl-treated tumors, n = 7 for siOIN1 #1-treated tumors). The graph in FIG. 4C shows the average value ± SD of the tumor weights. In FIG. 4C, * represents p < 0.05. Statistical analysis was performed by the Mann-Whitney U-test.

[0130] As shown in FIGS. 4A to 4C, it was shown that the injection of siOIN1 #1 significantly decreased the volume and weight of A2780-derived xenograft tumors.

[0131] In addition, the results of examining the expression of OIN1, RASSF5, and ADORA1 in tumors are shown in FIGS. 4D to 4F.

[0132] FIG. 4D shows the results of analyzing the knockdown efficiency of OIN1 by siOIN1 #1 injection using qRT-PCR. In FIG. 4D, the relative expression level of OIN1 was calculated by correcting with the expression level of GAPDH mRNA, and the graph shows the average value ± SD of the fold change in the expression level of OIN1 when compared with the siControl treatment condition (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.

[0133] FIGS. 4E and 4F show the results of analyzing 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 levels of each gene were calculated by correcting with the expression level of GAPDH mRNA, and the graph shows the average value ± SD of the fold change in the expression level of each gene when compared with the siControl treatment condition (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.

[0134] As shown in FIGS. 4D to 4F, it was confirmed that the expression of OIN1 in tumors decreased by injection of siOIN1 #1, while on the other hand, it was revealed that the expression of RASSF5 and ADORA1 increased.

[0135] From the above results, it was suggested that OIN1 controls the expression of RASSF5 and ADORA1 to suppress apoptosis and promotes 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 for female cancers.

[0136] <<OIN1 promotes the growth of endometrial cancer and breast cancer cells>> To explore the function of OIN1 in cancers other than ovarian cancer, we performed an OIN1 knockdown experiment using siRNA (siOIN1 #1 and siOIN1 #2) in endometrial cancer cell line Ishikawa.

[0137] Figure 5A shows the results of qRT-PCR analysis of OIN1 knockdown efficiency in Ishikawa treated with siOIN1 #1 or siOIN1 #2. In Figure 5A, the relative expression level of OIN1 was calculated by correcting it for the expression level of GAPDH mRNA. The graph shows the mean ± SD of the fold change in OIN1 expression compared to siControl treatment (n = 3). In Figure 5A, ** indicates p < 0.01. Statistical analysis was performed using two-way ANOVA.

[0138] As shown in Figure 5A, it was confirmed that treatment with siOIN1 #1 and siOIN1 #2 significantly reduced OIN1 expression in Ishikawa.

[0139] Figure 5B shows the results of a DNA assay that demonstrated that OIN1 knockdown inhibited Ishikawa proliferation. The graph in Figure 5B shows the mean ± SD of the measured DNA content (n = 5). In Figure 5B, *** indicates p < 0.0001. Statistical analysis was performed using two-way analysis of variance.

[0140] As shown in Figure 5B, it was confirmed that treatment with siOIN1 #1 and siOIN1 #2 significantly suppressed the proliferation of Ishikawa.

[0141] Next, we performed an OIN1 knockdown experiment using siOIN1 #1 in breast cancer cells, BrC-PDC.

[0142] The results of treating BrC-PDC with siOIN1 #1 and analyzing the knockdown efficiency of OIN1 by qRT-PCR are shown in Fig. 5C. In Fig. 5C, the relative expression level of OIN1 was calculated by correcting with the expression level of GAPDH mRNA, and the graph shows the average value ± SD of the change in the expression fold 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.

[0143] As shown in Fig. 5C, it was confirmed that the treatment with siOIN1 #1 significantly decreased the expression of OIN1 in BrC-PDC.

[0144] In addition, the results of examining whether the knockdown of OIN1 suppresses the growth of BrC-PDC by CellTiter-3D Cell Viability Assay (Promega) are shown in Fig. 5D. In Fig. 5D, ** represents p < 0.001. Statistical analysis was performed by Student's t-test.

[0145] As shown in Fig. 5D, it was confirmed that the treatment with siOIN1 #1 significantly suppressed the growth of BrC-PDC.

[0146] <<Overexpression of OIN1 promotes the growth of ovarian cancer cells and suppresses the expression of apoptosis-related genes>> Next, the effects of overexpression of OIN1 in ovarian cancer were analyzed.

[0147] The results of confirming the change in the expression of OIN1 RNA in A2780 and SKOV3 cells transfected with OIN1 expression plasmid or empty vector are shown in Fig. 6C (A2780 cells) and Fig. 6F (SKOV3 cells). The data are presented as mean ± SD (n = 3). In Figs. 6C and 6F, "Vector" indicates the result when transfected with the empty vector, and "OIN1" indicates the result when transfected with the OIN1 expression plasmid. Also, "***" in Figs. 6C and 6F indicates p < 0.001 (Student's t-test).

[0148] As shown in FIGS. 6C and 6F, it was confirmed by qRT-PCR that the expression of OIN1 was significantly increased by transfection of the OIN1 expression plasmid into A2780 and SKOV3 cells.

[0149] The results of analyzing the proliferation of A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or the empty vector using a DNA assay are shown in FIGS. 6A (A2780 cells) and 6B (SKOV3 cells). The data are presented as mean ± SD (n = 5). In FIGS. 6A and 6B, "Vector" indicates the result when transfected with the empty vector, and "OIN1" indicates the result when transfected with the OIN1 expression plasmid. Also, "*" in FIGS. 6A and 6B indicates p < 0.05, and "***" indicates p < 0.001 (Student's t-test).

[0150] As shown in FIGS. 6A and 6B, it was confirmed that overexpression of OIN1 significantly enhanced the proliferation of ovarian cancer cells.

[0151] The results of confirming the changes in the expression of RASSF5 mRNA and ADORA1 mRNA in A2780 and SKOV3 cells transfected with the OIN1 expression plasmid or the empty vector are shown in FIGS. 6D (A2780 cells, RASSF5), 6E (A2780 cells, ADORA1), 6G (SKOV3 cells, RASSF5), and 6H (SKOV3 cells, ADORA1). The data are presented as mean ± SD (n = 3). In FIGS. 6D, 6E, 6G, and 6H, "Vector" indicates the result when transfected with the empty vector, and "OIN1" indicates the result when transfected with the OIN1 expression plasmid. Also, "*" in FIGS. 6D, 6E, 6G, and 6H indicates p < 0.05, and "**" indicates p < 0.01 (Student's t-test).

[0152] As shown in FIGS. 6D, 6E, 6G, and 6H, it was confirmed that overexpression of OIN1 decreased the expression of RASSF5 and ADORA1 in A2780 cells and SKOV3 cells.

[0153] <<OIN1 is highly expressed in endometrial cancer>> The expression level of OIN1 was calculated as RPKM (reads per kilobase per million sequenced reads) from RNA sequencing analysis of 34 endometrial cancer specimens. The results are shown in Figure 7.

[0154] As shown in Figure 7, it was demonstrated that there were endometrial cancer specimens that expressed OIN1 at levels comparable to those of ovarian cancer specimens.

[0155] These results indicate that OIN1 is important not only for ovarian cancer, but also for the growth of other female cancers such as breast cancer and endometrial cancer, an example of uterine cancer, and suggest that OIN1 is a promising tumor marker and a target for the diagnosis and treatment of female cancers. Furthermore, it was shown that the double-stranded nucleic acid molecule of the present invention can suppress the proliferation of not only ovarian cancer cells but also female cancer cells such as breast cancer cells and uterine cancer cells, suggesting that double-stranded nucleic acid molecules targeting OIN1 may be applicable to nucleic acid drug discovery for female cancers.

[0156] The present invention includes, for example, the following aspects. <1> A double-stranded nucleic acid molecule for inhibiting expression of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1, (a) a sense strand containing a nucleotide sequence corresponding to a target sequence consisting of a nucleotide 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 comprising a base sequence complementary to the sense strand, which forms a double strand. <2> The above-mentioned compound is either a double-stranded RNA or a double-stranded RNA-DNA chimera. <1> It is a double-stranded nucleic acid molecule described in <3> The above-mentioned siRNA or chimeric siRNA <1> from <2> The double-stranded nucleic acid molecule according to any one of the above items. <4> The above-mentioned siRNA <1> from <3> The double-stranded nucleic acid molecule according to any one of the above items. <5> The aforementioned <1> from <4> The DNA is characterized by comprising a base sequence encoding any one of the double-stranded nucleic acid molecules described above. <6> The aforementioned <5> A vector characterized by comprising the DNA described in <7> The aforementioned <1> from <4> The double-stranded nucleic acid molecule according to any one of <5> and the DNA described in <6> and a female cancer cell proliferation inhibitor characterized by comprising at least one of the vectors described above. <8> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <7> It is a female cancer cell proliferation inhibitor described in . <9> In female cancer cells, <7> from <8> The present invention relates to a method for inhibiting the proliferation of female cancer cells, characterized by applying the female cancer cell proliferation inhibitor described in any one of the above. <10> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <9> This is a method for inhibiting the proliferation of female cancer cells described in the above. <11> The aforementioned <1> from <4> The double-stranded nucleic acid molecule according to any one of <5> and the DNA described in <6> and a tumor formation inhibitor for female cancer, characterized by comprising at least one of the vectors described above. <12> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <11> The present invention is a tumor formation inhibitor for female cancers described in . <13> For female cancer tumors, <11> from <12> The present invention relates to a method for inhibiting tumor formation in female cancer, which comprises applying the agent for inhibiting tumor formation in female cancer described in any one of the above. <14> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <13> This is a method for suppressing tumor formation in female cancers described in the above. <15> A pharmaceutical for preventing or treating female cancer, comprising: <7> from <8> The female cancer cell proliferation inhibitor according to any one of the preceding claims and <11> from <12> The present invention relates to a pharmaceutical composition comprising at least one of the tumor formation inhibitors for female cancers described above. <16> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <15> It is a medicine described in <17> To the individual, <15> from <16> The present invention relates to a method for preventing or treating female cancer, which comprises administering the pharmaceutical agent described in any one of the above. <18> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <17> The present invention relates to a method for preventing or treating female cancer. <19> This is a method for evaluating female cancer, characterized by comprising evaluating whether a subject is suffering from female cancer or has the potential to be suffering from female cancer, using the presence or expression level of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample derived from the subject as an indicator. <20> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <19> This is a method for evaluating female cancers described in <21> This is a marker for evaluating female cancer, characterized by containing a long non-coding RNA consisting of the base sequence represented by SEQ ID NO:1. <22> The female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer. <21> It is a marker for evaluating female cancers described in< / dna>

Claims

1. An siRNA for suppressing expression of a long non-coding RNA consisting of the 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) siRNA comprising the sense strand of (a) and an antisense strand comprising a base sequence complementary to the sense strand, which forms a double strand with the sense strand.

2. A DNA comprising a base sequence encoding the siRNA of claim 1.

3. A vector comprising the DNA of claim 2.

4. The method comprises at least one of the siRNA according to claim 1, the DNA according to claim 2, and the vector according to claim 3, 1. A female cancer cell proliferation inhibitor characterized by being used to inhibit the proliferation of at least one female cancer cell, including ovarian cancer cells, uterine cancer cells, and breast cancer cells.

5. The method comprises at least one of the siRNA according to claim 1, the DNA according to claim 2, and the vector according to claim 3, 1. A female cancer tumorigenesis inhibitor characterized by being used to inhibit tumorigenesis of at least one female cancer selected from ovarian cancer, uterine cancer, and breast cancer.

6. A pharmaceutical for preventing or treating at least one of female cancers, including ovarian cancer, uterine cancer, and breast cancer, characterized in that it contains at least one of the female cancer cell proliferation inhibitor described in claim 4 and the female cancer tumor formation inhibitor described in claim 5.

7. A method for assisting in the evaluation of female cancer, comprising assisting in the evaluation of whether or not a subject has female cancer or has a possibility of having female cancer, using as an indicator the presence or absence or expression level of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 in a sample derived from the subject, A method for assisting in the evaluation of female cancer, wherein the female cancer is at least one of ovarian cancer, uterine cancer, and breast cancer.

8. Use of a long non-coding RNA consisting of the base sequence represented by SEQ ID NO: 1 as an auxiliary marker for evaluating female cancer, to assist in assessing whether a subject is suffering from or is likely to suffer from at least one of female cancers including ovarian cancer, uterine cancer, and breast cancer.

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