WO2022044788A1 - Molécule d'acide nucléique double brin, adn, vecteur, agent d'inhibition de croissance de cellule cancéreuse femelle, agent d'inhibition de formation de tumeur cancéreuse femelle, produit pharmaceutique et utilisation d'arn non codant à longue chaîne - Google Patents

Molécule d'acide nucléique double brin, adn, vecteur, agent d'inhibition de croissance de cellule cancéreuse femelle, agent d'inhibition de formation de tumeur cancéreuse femelle, produit pharmaceutique et utilisation d'arn non codant à longue chaîne 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)
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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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学校法人埼玉医科大学
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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

L'invention concerne une molécule d'acide nucléique double brin destinée à inhiber l'expression d'un ARN non codant à longue chaîne qui comprend une séquence de bases représentée par SEQ ID NO : 1 et qui comprend les éléments suivants : (a) un brin sens qui comprend une séquence de bases qui correspond à une séquence cible comprenant une séquence de bases représentée par SEQ ID NO : 2 ou SEQ ID NO : 3 ; et (b) un brin antisens qui comprend une séquence de bases qui est complémentaire du brin sens (a) et qui forme, avec le brin sens, un double brin.
PCT/JP2021/029473 2020-08-28 2021-08-10 Molécule d'acide nucléique double brin, adn, vecteur, agent d'inhibition de croissance de cellule cancéreuse femelle, agent d'inhibition de formation de tumeur cancéreuse femelle, produit pharmaceutique et utilisation d'arn non codant à longue chaîne WO2022044788A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
RUAN ZHENGYI, ZHAO DONG: "Long intergenic noncoding RNA LINC00284 knockdown reduces angiogenesis in ovarian cancer cells via up‐regulation of MEST through NF‐κB1", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 33, no. 11, 1 November 2019 (2019-11-01), US, pages 12047 - 12059, XP055905352, ISSN: 0892-6638, DOI: 10.1096/fj.201900101RR *
XU HUI, ZHENG JING-FANG, HOU CONG-ZHE, LI YUE, LIU PEI-SHU: "Up-regulation of long intergenic noncoding RNA 01296 in ovarian cancer impacts invasion, apoptosis and cell cycle distribution via regulating EMT", CELLULAR SIGNALLING., ELSEVIER SCIENCE LTD., GB, vol. 62, 1 October 2019 (2019-10-01), GB , pages 109341, XP055905349, ISSN: 0898-6568, DOI: 10.1016/j.cellsig.2019.06.006 *
ZHANG CHU; LIU JIE; ZHANG YANG; LUO CHENGYAN; ZHU TONG; ZHANG RONGRONG; YAO RUIQIN: "LINC01210 accelerates proliferation, invasion and migration in ovarian cancer through epigenetically downregulating KLF4", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 119, 9 September 2019 (2019-09-09), FR , XP085848604, ISSN: 0753-3322, DOI: 10.1016/j.biopha.2019.109431 *
ZHANG, RUI ET AL: "LncRNAs and cancer ", ONCOLOGY LETTERS, vol. 12, no. 2, 1 January 2016 (2016-01-01), pages 1233 - 1239, XP009534515, DOI: 10.3892/ol.2016.4770 *

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