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WO2019100618A1 - 微小rna及其在制备抗肿瘤药物中的应用 - Google Patents

微小rna及其在制备抗肿瘤药物中的应用 Download PDF

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WO2019100618A1
WO2019100618A1 PCT/CN2018/077481 CN2018077481W WO2019100618A1 WO 2019100618 A1 WO2019100618 A1 WO 2019100618A1 CN 2018077481 W CN2018077481 W CN 2018077481W WO 2019100618 A1 WO2019100618 A1 WO 2019100618A1
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seq
cancer
rna molecule
set forth
tumor
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French (fr)
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汪维鹏
孟凡义
邱雅静
孔洁红
陈喜华
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苏州大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention relates to the field of biopharmaceutical technology, in particular to microRNAs and their use in preparing antitumor drugs.
  • Effective anti-tumor immunotherapy relies on efficient activation of cytotoxic T lymphocytes (CTLs), whereas efficient activation and functional mediated T-cells are dependent on the synergy provided by antigen-presenting cells and ligand/receptor pairs on the surface of T cells.
  • Stimulus signal is the most basic co-stimulation signal recognized so far, including B7-1, B7-2, B7h, PD-L1 (B7-H1), B7-H2, B7-H3, B7-H4, etc.
  • PD-L1, B7-H3 and B7-H4 are important members of the B7 family of costimulatory molecules discovered in recent years. They are found to be abnormally highly expressed in various tumor tissues, including intestinal cancer, gastric cancer, esophageal cancer, lung cancer, and urinary tract. Metastatic cell carcinoma, renal cancer, prostate cancer, etc.; and its high expression is significantly associated with poor prognosis. A large number of studies have also confirmed that good anti-tumor effects can be obtained by inhibiting the expression of PD-L1, B7-H3 or B7-H4; currently, the PD-L1 monoclonal antibody drug already marketed has Roche's Atezolizumab for the treatment of urine.
  • MicroRNAs are a class of endogenous non-coding small RNAs of about 22 nucleotides long found in eukaryotic cells in recent years, with 5'-end 2-9 bases. Protein binding can be inhibited at the translational level by binding to the 3'-UTR of the target gene, thereby exerting an important regulatory role in gene expression.
  • miRBase microRNAs
  • each microRNA may be involved in the regulation of translation of 100 to 200 target genes.
  • MicroRNAs participate in the life processes of cell growth, differentiation, proliferation and apoptosis due to their extensive regulation, affecting almost all signaling pathways, and participating in various physiological and pathological processes, especially in the occurrence and development of tumors. Extremely important role.
  • microRNAs are abnormally expressed in tumors, and some of them have increased and decreased expression of microRNAs, which play a role in promoting cancer or suppressing cancer by inhibiting the expression of tumor suppressor genes or up-regulating the expression of oncogenes.
  • Clain et al found that two microRNA genes, miR-15 and miR-16, are frequently deleted in patients with chronic lymphocytic leukemia, revealing for the first time the close relationship between microRNAs and tumors. Later, more and more microRNAs were found to be abnormally expressed in tumors, such as low expression of miR-34a-5p, miR-143, miR-145 and highly expressed miR-21, miR-27a, miR-155, etc. .
  • microRNA-expressing mimics mimic and agomir, etc.
  • inhibitors that express microRNAs inhibitors that express microRNAs (inhibitor and antagomir, etc.). Transfer to exert anti-tumor effects.
  • miR-34a-5p mimic amphotericin liposome preparation MRX34. Since miR-34a-5p has shown excellent anti-tumor activity and safety in cell and animal levels, Mirna Therapeutic USA promoted a multi-center Phase I clinical trial in 2013 for the treatment of primary disease. Patients with liver cancer, small cell lung cancer, lymphoma, melanoma, multiple myeloma or renal cell carcinoma have also become the first microRNA drugs to enter clinical trials.
  • RG-101 N-acetyl-D-galactosamine-modified anti-miR-122 nucleic acid fragment
  • RG-012 miR-21 inhibitor
  • RG-125/AZD4076 N-acetylgalactosamine modified MicroRNA drugs such as miR-103/107 inhibitory molecule
  • MRG-201 miR-29mimic
  • MRG-106 antiimiR-155 locked nucleotide
  • the technical problem to be solved by the present invention is to provide microRNAs and their use in the preparation of antitumor drugs.
  • the microRNA provided by the invention has good anti-tumor effect and does not cause abnormal secretion of cytokines.
  • the present invention provides an RNA molecule in which any one of the 10th to 22nd positions at the 5' end of the miR-34a-5p sequence is mutated to n, and the n is A, C, G or U.
  • the invention also provides RNA molecules that are fully complementary or partially complementary to the RNA molecule sequences described above.
  • the partially complementary sticky ends are 2 bp.
  • the sticky end is located at the 3' end of the complementary RNA molecule.
  • RNA molecule obtained by modifying any one or more bases in the RNA molecule provided by the invention.
  • RNA molecule provided by the present invention for the preparation of a medicament for inhibiting protein levels of PD-L1, B7-H3, B7-H4 and the like.
  • RNA molecule provided by the present invention for the preparation of a medicament for inhibiting the growth and/or proliferation of tumor cells.
  • RNA molecule provided by the invention for preparing a therapeutic anti-tumor drug.
  • the tumor is astrocytoma, anaplastic large cell lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, angiosarcoma, breast cancer, B cell lymphoma, bladder cancer, cervical cancer, head and neck cancer, chronic lymphocytes Leukemia, chronic myelogenous leukemia, colon cancer, rectal cancer, endometrial cancer, glioma, glioblastoma, gastric cancer, gastrinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin's lymph Tumor, Kaposi's sarcoma leukemia, lung cancer, leiomyosarcoma, laryngeal squamous cell carcinoma, melanoma, mucosa-associated lymphoid tissue B-cell lymphoma, medulloblastoma, mantle cell lymphoma, meningioma, myeloid leukemia, multiple Myeloma, high-risk mye
  • the invention also provides an antitumor drug, comprising a living chain and a complementary strand;
  • the active strand is an RNA molecule or a modified RNA molecule, and the RNA molecule is mutated to n from any of the 10th to 22nd positions of the 5' end of the miR-34a-5p sequence, and the n is A. , C, G or U; the complementary strand is complementary to the active strand.
  • RNA molecule provided by the present invention is mutated to any of the 10th to 22nd positions of the 5' end of the miR-34a-5p sequence to n, and the n is A, C, G or U; Its complementary strand can inhibit the protein levels of PD-L1, B7-H3, B7-H4 and other proteins in tumor cells, inhibit tumor cell growth and/or proliferation, and thus play an anti-tumor effect. Moreover, studies have shown that the microRNAs provided by the present invention have no significant effect on the secretion of cytokines in serum while anti-tumor.
  • Figure 1 shows the results of the ability of miR-449a and miR-34a-5p mimics to inhibit tumor cell growth; wherein, Figure 1-a shows the inhibitory effect on HCT-116 intestinal cancer cells; Figure 1-b shows the HCT-8 The inhibitory effect of intestinal cancer cells, Figure 1-c shows the inhibitory effect on Caco-2 intestinal cancer cells; Figure 1-d shows the inhibitory effect on SW480 intestinal cancer cells; Figure 1-e shows the PANC-1 pancreatic cancer cells Inhibition effect; and Figure 1-f shows the inhibitory effect on Hela cervical cancer cells; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 2 shows the microRNA (MIR1A, MIR2G, MIR3A, MIR4C, MIR5U, MIR6U, MIR7A, MIR8A, MIR9C, MIR10C, MIR11A, MIR12U and MIR13A) mimics and miR-34a-5p treated HCT in the examples of the present invention.
  • -116 results of relative growth ability of colorectal cancer cells; *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
  • Figure 3 shows the results of detection of the ability of microRNAs (MIR1A and MIR9C) and miR-34a-5p mimics to inhibit tumor cell growth in an embodiment of the present invention
  • Figure 3-a shows inhibition of HCT-116 intestinal cancer cells
  • Fig. 3-b shows the inhibitory effect on HCT-8 intestinal cancer cells
  • Fig. 3-c shows the inhibitory effect on Caco-2 intestinal cancer cells
  • Fig. 3-d shows the inhibitory effect on SW480 intestinal cancer cells
  • Fig. 3 -e shows inhibitory effect on SGC-7901 gastric cancer cells
  • Fig. 3-f shows inhibitory effect on A549 lung cancer cells
  • Fig. 3-g shows inhibitory effect on PANC-1 pancreatic cancer cells
  • Fig. 3-h shows Hela Inhibition effect of cervical cancer cells
  • MIR1A and MIR9C miRNAs
  • Figure 4 shows the ability of different concentrations (0.1, 2.0, 10, 25, 50, 100 nM) of microRNAs (MIR1A and MIR9C) and miR-34a-5p mimics to inhibit the growth of HCT-116 tumor cells in an embodiment of the invention.
  • the results of the assay wherein, Figure 4-a shows the inhibitory effect of miR-34a-5p; Figure 4-b shows the inhibitory effect of MIR1A, and Figure 4-c shows the inhibitory effect of MIR9C;
  • Figure 5 shows the results of the ability of the blunt-end or cohesive-end microRNAs (MIR1A and MIR9C) and miR-34a-5p mimics to inhibit the growth of HCT-116 tumor cells in the examples of the present invention; wherein, Figure 5-a shows the blunt end The inhibitory effect of miR-34a-5p on the sticky end miR-34a-5p; Figure 5-b shows the inhibitory effect of the blunt-end MIR1A and the sticky end MIR1A, and Figure 5-c shows the inhibitory effect of the blunt-ended MIR9C and the sticky end MIR9C;
  • Figure 6 shows the results of the detection of the ability of microRNA (MIR9CM) and miR-34a-5p agonist to inhibit the growth of HCT-116 cells in nude mice xenografts in the examples of the present invention; wherein, Figure 6-a shows the growth curve of the transplanted tumor Figure 6-b shows the change in body weight of nude mice bearing tumors; Figure 6-c shows the change in food intake of nude mice bearing tumors;
  • Figure 7 shows the results of flow cytometry detection of cytokine secretion levels in HCT-116 cells treated with microRNA (MIR9C) mimetic in the examples of the present invention; wherein, Figure 7-a shows the results of IL-2 protein detection.
  • Figure 7-b shows the results of IL-4 protein assay;
  • Figure 7-c shows the results of IL-6 protein assay;
  • Figure 7-d shows the results of IL-10 protein assay;
  • Figure 7-e shows the results of TNF-alpha protein The test results;
  • Figure 7-f shows the detection results of IFN- ⁇ protein;
  • Figure 8 shows the results of flow cytometry detection of cytokine secretion levels in a HCT-116 cell/T lymphocyte co-culture system treated with microRNA (MIR9C) mimetic in the examples of the present invention
  • Figure 8-a shows The results of IL-2 protein detection
  • Figure 8-b shows the results of IL-4 protein
  • Figure 8-c shows the results of IL-6 protein
  • Figure 8-d shows the results of IL-10 protein
  • e shows the detection result of TNF- ⁇ protein
  • FIG. 8-f shows the detection result of IFN- ⁇ protein
  • Figure 9 shows the results of flow cytometry detection of cytokine secretion levels in serum of C57BL/6 black mice treated with microRNA (MIR9CM) agonist in the examples of the present invention
  • Figure 9-a shows IL-2 protein The results of the test
  • Figure 9-b shows the results of IL-4 protein
  • Figure 9-c shows the results of IL-6 protein
  • Figure 9-d shows the results of IL-10 protein
  • Figure 9-e shows TNF- The detection result of ⁇ protein
  • FIG. 9-f shows the detection result of IFN- ⁇ protein
  • Figure 10 shows the results of Western blot detection of PD-L1, B7-H3 and B7-H4 proteins in HCT-116 cells treated with microRNAs (MIR1A and MIR9C) and miR-34a-5p mimics in the examples of the present invention
  • Figure 11 shows the results of Western blot detection of protein expression in HCT-116 cell xenografts treated with microRNA (MIR9CM) agonists in the examples of the present invention; wherein, Figure 11-a shows the results of detection of PD-L1 protein; 11-b shows the detection result of B7-H3 protein; Fig. 11-c shows the detection result of B7-H4 protein.
  • MIR9CM microRNA
  • the present invention provides microRNAs and their use in the preparation of antitumor drugs, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application of the present invention may be modified or combined and modified to achieve and apply the present invention without departing from the scope of the present invention. Invention technology.
  • RNA molecule provided by the present invention is a microRNA (or miRNA), which refers to a single-stranded oligoribonucleic acid.
  • a ribonucleotide is a chain-like molecule formed by condensation of a nucleotide via a phosphodiester bond.
  • a ribonucleotide molecule consists of one molecule of base, one molecule of ribose, and phosphoric acid.
  • the miRNA provided by the present invention has four kinds of bases, A adenine, G guanine, C cytosine, U uracil, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, Astragalus, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-sulfur Other alkynyl derivatives of uracil and cytosine, 5-propynyl uracil and pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-sulfur Uracil, 8-halo, 8-amino, 8-indenyl, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-
  • the base may be an unmodified base or a modified base.
  • the modified base refers to a base linking group including, but not limited to, NH 2 , biotin, amine, lower aminoalkyl, lower alkyl, NHCOCH 3 , acetyl, 2′-oxy-methyl (2'O-Me), DMTO, fluorescein, thiol or acridine.
  • the modified base refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of the RNA molecular chain or its complementary strand provided by the present invention. , 16, 17, 18, 19, 20, 21 or 22 bases are attached to the modifying group.
  • the modification refers to the connection of any one or more groups or a combination thereof to the base of the RNA molecular chain or the complementary strand thereof provided by the present invention.
  • the ribose may be an unmodified ribose or a modified ribose.
  • the modified ribose refers to a ribose linking group including, but not limited to, lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkylaryl or O-aralkyl, SH, SCH 3 , Cl, Br, CN, OCN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocyclic aminoalkyl, aminoalkyl, poly An aminoalkyl group, a substituted silyl group, an RNA cleavage group, an intercalator, a group for improving the pharmacokinetic properties of microRNAs, or a group for improving the pharmacodynamic properties of microRNAs, and having similarities Other substituents of nature.
  • the modified ribose refers to any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of the RNA molecular chain or its complementary strand provided by the present invention. , 16, 17, 18, 19, 20, 21 or 22 ribose linkage modifying groups.
  • the modification refers to a ribose linkage in the RNA molecule chain or a complementary strand thereof provided by the present invention to any one or more groups or a combination thereof.
  • the complementation also known as complementary pairing, refers to the attachment of a base to its complementary base by hydrogen bonding.
  • the complementary base pair A forms two pairs of hydrogen bonds between U, and three pairs of hydrogen bonds are formed between C and G.
  • the complete complementation means that the sequences are perfectly matched and do not form sticky ends.
  • the partial complementation means that the sequences are perfectly matched but form a sticky end.
  • the mutation of the present invention means that one base is substituted by another base, and after the mutation, the number of bases in the RNA molecule does not change.
  • the mutation does not have a base substituted with the same base as itself, that is, the mutation does not exist in which A is substituted by A, C is substituted by C, G is substituted by G, and U is substituted by U.
  • RNA molecule provided by the present invention is mutated to any one of the 10th to 22nd positions at the 5' end of the miR-34a sequence, and the n is A, C, G or U.
  • the invention also provides RNA molecules that are fully complementary or partially complementary to the RNA molecule sequences described above.
  • the partially complementary sticky ends are 2 bp.
  • the sticky end is located at the 3' end of the complementary RNA molecule.
  • the mutated miR-34a-5p is provided as a living chain, and an RNA molecule which is completely complementary or partially complementary thereto is referred to as a complementary strand.
  • the miR-34a-5p sequence is set forth in SEQ ID NO.
  • the fully complementary microRNA molecule pair comprises the active strand of the sequence set forth in SEQ ID NO: 1 and the complementary strand of the sequence set forth in SEQ ID NO. 27; or comprises the sequence set forth in SEQ ID NO: 17.
  • Partially complementary pairs of microRNA molecules include:
  • the active strand sequence is shown in SEQ ID NO. 1, and the complementary strand sequence is shown in SEQ ID NO.
  • SEQ ID NO. 1 is the mutation of the 10th base C at the 5' end of the miR-34a-5p sequence to n, and the n is A, G or U.
  • n in SEQ ID NO. 1 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 1 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 2; the 21st to 22nd positions of the 5' end of SEQ ID NO. 1 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 2 form a sticky end.
  • the active strand sequence is shown in SEQ ID NO. 3, and the complementary strand sequence thereof is shown in SEQ ID NO.
  • SEQ ID NO. 3 is the mutation of the 11th base U at the 5' end of the miR-34a-5p sequence to n, and the n is A, C or G.
  • n in SEQ ID NO. 3 is G.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 3 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 4; the 21st to 22nd positions of the 5' end of SEQ ID NO. 3 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 4 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 5, and the complementary strand sequence thereof is set forth in SEQ ID NO.
  • SEQ ID NO. 5 is the mutation of base 12 of the 5' end of the miR-34a-5p sequence to N, and the n is A, C or G.
  • n in SEQ ID NO. 5 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 5 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 6; the 21st to 22nd positions of the 5' end of SEQ ID NO. 5 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 6 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 7, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 7 is the mutation of base 13 at the 5' end of the miR-34a-5p sequence to n, and the n is C, G or U. In a specific embodiment, n in the SEQ ID NO. 7 is C.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 7 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 8; the 21st to 22nd positions of the 5' end of SEQ ID NO. 7 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 8 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 9, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 9 is the mutation of the 14th base G at the 5' end of the miR-34a-5p sequence to n, and the n is A, C or U. In a particular embodiment, n described in SEQ ID NO. 9 is U.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 9 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 10; the 21st to 22nd positions of the 5' end of SEQ ID NO. 9 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 10 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 11, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 11 is the mutation of the 15th base C at the 5' end of the miR-34a-5p sequence to n, and the n is A, G or U. In a particular embodiment, n in SEQ ID NO. 11 is U.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 11 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 12; the 21st to 22nd positions of the 5' end of SEQ ID NO. 11 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 12 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 13, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 13 is a mutation in the 16th base 5 of the 5' end of the miR-34a-Sp sequence to n, and the n is A, C or G.
  • n in SEQ ID NO. 13 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 13 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 14; the 21st to 22nd positions of the 5' end of SEQ ID NO. 13 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 14 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 15, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 15 is a mutation in the 17th base of the 5' end of the miR-34a-5p sequence to N, and the n is A, C or U. In a particular embodiment, n in SEQ ID NO. 15 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 15 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 16: the 21st to 22nd positions of the 5' end of SEQ ID NO. 15 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 16 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 17, and the complementary strand sequence is set forth in SEQ ID NO.
  • SEQ ID NO. 17 is the mutation of base 18 at the 5' end of the miR-34a-5p sequence to n, and the n is A, C or U.
  • n described in SEQ ID NO. 17 is C.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 17 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 18; the 21st to 22nd positions of the 5' end of SEQ ID NO. 17 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 18 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 19, and the complementary strand sequence thereof is set forth in SEQ ID NO.
  • SEQ ID NO. 19 is a mutation in the 19th base 5 of the 5' end of the miR-34a-5p sequence to n, and the n is A, C or G. In a particular embodiment, n in the SEQ ID NO. 19 is C.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 19 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 20; the 21st to 22nd positions of the 5' end of SEQ ID NO. 19 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 20 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 21, and the complementary strand sequence thereof is set forth in SEQ ID NO.
  • SEQ ID NO. 21 is the mutation of base 20 at the 5' end of the miR-34a-5p sequence to N, and the n is A, C or G. In a particular embodiment, n in SEQ ID NO. 21 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 21 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 22; the 21st to 22nd positions of the 5' end of SEQ ID NO. 21 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 22 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 23, and the complementary strand sequence thereof is set forth in SEQ ID NO.
  • SEQ ID NO. 23 is the mutation of the 21st base G at the 5' end of the miR-34a-5p sequence to n, and the n is A, C or U. In a particular embodiment, n described in SEQ ID NO. 23 is U.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 23 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 24; the 21st to 22nd positions of the 5' end of SEQ ID NO. 23 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 24 form a sticky end.
  • the active strand sequence is set forth in SEQ ID NO. 25, and the complementary strand sequence thereof is set forth in SEQ ID NO.
  • SEQ ID NO. 25 is the mutation of the 22nd base U at the 5' end of the miR-34a-5p sequence to n, and the n is A, C or G.
  • n in SEQ ID NO. 25 is A.
  • the 1st to 20th positions of the 5' end of SEQ ID NO. 25 are complementary to the 1st to 20th positions of the 5' end of SEQ ID NO: 24; the 21st to 22nd positions of the 5' end of SEQ ID NO. 25 form a sticky end; The 21st to 22nd positions of the 5' end of ID NO. 24 form a sticky end.
  • RNA molecule obtained by modifying any one or more bases in the RNA molecule provided by the invention.
  • the complementary strand is modified.
  • the modification comprises: linking a 2'-oxy-methyl group to any one or more ribose in the sequence, replacing a hydroxyl group in any one or more of the phosphate groups with sulfur, and making the 3'-end ribose The upper hydroxyl group is linked to cholesterol.
  • RNA molecule provided by the present invention for the preparation of a medicament for inhibiting the growth and/or proliferation of tumor cells.
  • microRNA molecules provided by the present invention have significant inhibitory effects on the growth of various tumor cells, and show that the microRNA molecules provided by the present invention have a good broad-spectrum antitumor activity; and the results show that The microRNA molecules provided by the invention have superior inhibitory effects on tumor cells than miR-34a-5p.
  • the tumor cells to be tested in the present invention include colorectal cancer cells, gastric cancer cells, lung cancer cells, pancreatic cancer cells, and/or cervical cancer cells.
  • the experimental tumor cells were HCT-116 colorectal cancer cells, HCT-8 colorectal cancer cells, Caco-2 colorectal cancer cells, SW480 colorectal cancer cells, SGC-7901 gastric cancer cells, A549 lung cancer cells, PANC. -1 Antitumor activity in tumor cells such as pancreatic cancer cells and Hela cervical cancer cells.
  • microRNA molecule pairs tested some of the inhibitory activities against tumor cell growth were comparable to those of miR-34a-5p, and the sequences of the active strands in these microRNA pairs were as follows: SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO.
  • these molecular pairs are:
  • MIR1A the active strand sequence is set forth in SEQ ID NO. 1, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 2, wherein n is U;
  • MIR6U the active strand sequence is set forth in SEQ ID NO. 11, wherein n is U, and the complementary strand sequence is set forth in SEQ ID NO. 12, wherein n is A;
  • MIR9C the active strand sequence is set forth in SEQ ID NO. 17, wherein n is C and the complementary strand sequence is set forth in SEQ ID NO. 18, wherein n is G;
  • MIR10C the active strand sequence is set forth in SEQ ID NO. 19, wherein n is C and the complementary strand sequence is set forth in SEQ ID NO. 20, wherein n is G;
  • MIR11A The active strand sequence is set forth in SEQ ID NO. 21, wherein n is A and the complementary strand sequence is set forth in SEQ ID NO. 22, wherein n is U.
  • microRNA molecule pair has an inhibitory activity against tumor cell growth comparable to that of miR-34a-5p, but they have higher drug safety relative to miR-34a-5p.
  • MIR9C had no significant effect on the secretion of five cytokines such as IL-4, IL-6, IL-10, IFN- ⁇ and TNF- ⁇ in the serum of experimental mice.
  • microRNA pairs tested some of the inhibitory activities against tumor cell growth were significantly better than miR-34a-5p, p ⁇ 0.05, and the sequences of the active strands in these microRNA pairs were as follows: SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 23, SEQ ID NO.
  • these molecular pairs are:
  • MIR2G the active strand sequence is set forth in SEQ ID NO. 3, wherein n is G; the complementary strand sequence is set forth in SEQ ID NO. 4, wherein n is C;
  • MIR3A the active strand sequence is set forth in SEQ ID NO. 5, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 6, wherein n is U;
  • MIR4C the active strand sequence is set forth in SEQ ID NO. 7, wherein n is C; the complementary strand sequence is set forth in SEQ ID NO. 8, wherein n is G;
  • MIR5U the active strand sequence is set forth in SEQ ID NO. 9, wherein n is U; the complementary strand sequence is set forth in SEQ ID NO. 10, wherein n is A;
  • MIR7A the active chain sequence is set forth in SEQ ID NO. 13, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 14, wherein n is U;
  • MIR8A the active chain sequence is set forth in SEQ ID NO. 15, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 16, wherein n is U;
  • MIR12U the active strand sequence is set forth in SEQ ID NO. 23, wherein n is U; the complementary strand sequence is set forth in SEQ ID NO.
  • MIR13A The active strand sequence is set forth in SEQ ID NO. 25, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO.
  • RNA molecule provided by the present invention for the preparation of a medicament for inhibiting protein levels of PD-L1, B7-H3, B7-H4 and the like.
  • the present invention measures the effect of pairs of microRNA molecules on the expression of negative costimulatory molecules such as PD-L1, B7-H3 and B7-H4. It was confirmed by experiments that the microRNA molecule provided by the present invention significantly inhibited the expression of PD-L1, B7-H3 and B7-H4 proteins in HCT-116 cells.
  • the active strand sequence in the pair of microRNA molecules used to determine expression of a negative costimulatory molecule is SEQ ID NO. 1 or SEQ ID NO.
  • these molecular pairs are:
  • MIR1A the active strand sequence is set forth in SEQ ID NO. 1, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 2, wherein n is U;
  • MIR9C the active strand sequence is set forth in SEQ ID NO. 17, wherein n is C and the complementary strand sequence is set forth in SEQ ID NO. 18, wherein n is G;
  • Tumor models were constructed by inoculating HCT-116 cells in nude mice. After the tumor is grown to 150-200 mm 3 , the microRNA provided by the present invention is administered for treatment. The results showed that microRNA significantly inhibited the growth of HCT-116 cells in nude mice, but did not affect the body weight and food intake of the mice. Moreover, the tumor volume provided by the microRNA treatment group was significantly smaller than miR when the drug was just stopped. -34a-5p group.
  • RNA molecule provided by the invention for preparing a therapeutic anti-tumor drug.
  • the sequence of the active strand is as shown in SEQ ID NO. 17, wherein n is C; the sequence of the complementary strand is as shown in SEQ ID NO. 18, wherein n is G.
  • each ribose molecule in SEQ ID NO. 18 is linked to a 2'-oxy-methyl group, and the 1, 2, 19, 20, 21 and/or 22 phosphate groups at the 5' end.
  • the hydroxyl group in the middle is replaced by sulfur, and the hydroxyl group on the 3' terminal ribose is linked to cholesterol.
  • the tumor is astrocytoma, anaplastic large cell lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, angiosarcoma, breast cancer, B cell lymphoma, bladder cancer, cervical cancer, head and neck cancer, chronic lymphocytes Leukemia, chronic myelogenous leukemia, colon cancer, rectal cancer, endometrial cancer, glioma, glioblastoma, gastric cancer, gastrinoma, hepatoblastoma, hepatocellular carcinoma, Hodgkin's lymph Tumor, Kaposi's sarcoma leukemia, lung cancer, leiomyosarcoma, laryngeal squamous cell carcinoma, melanoma, mucosa-associated lymphoid tissue B-cell lymphoma, medulloblastoma, mantle cell lymphoma, meningioma, myeloid leukemia, multiple Myeloma, high-risk mye
  • the present invention further validates the antitumor activity of fully complementary microRNA molecule pairs and partially complementary microRNA molecule pairs.
  • the results showed that the anti-tumor activity of all cohesive terminal microRNAs was significantly stronger than the corresponding blunt-ended microRNAs, indicating that the cohesive terminal microRNAs have stronger anti-tumor activity.
  • the invention also provides an antitumor drug, comprising a living chain and a complementary strand;
  • the active strand is an RNA molecule or a modified RNA molecule, and the RNA molecule is mutated to n from any of the 10th to 22nd positions of the 5' end of the miR-34a-5p sequence, and the n is A. , C, G or U; the complementary strand is complementary to the active strand.
  • the anti-tumor drug comprises the active strand sequence: SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO.
  • microRNA molecules Specifically, the following pairs of microRNA molecules are included:
  • MIR1A the active strand sequence is set forth in SEQ ID NO. 1, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 2, wherein n is U;
  • MIR2G the active strand sequence is set forth in SEQ ID NO. 3, wherein n is G; the complementary strand sequence is set forth in SEQ ID NO. 4, wherein n is C;
  • MIR3A the active strand sequence is set forth in SEQ ID NO. 5, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 6, wherein n is U;
  • MIR4C the active strand sequence is set forth in SEQ ID NO. 7, wherein n is C; the complementary strand sequence is set forth in SEQ ID NO. 8, wherein n is G;
  • MIR5U the active strand sequence is set forth in SEQ ID NO. 9, wherein n is U; the complementary strand sequence is set forth in SEQ ID NO. 10, wherein n is A;
  • MIR6U the active strand sequence is set forth in SEQ ID NO. 11, wherein n is U, and the complementary strand sequence is set forth in SEQ ID NO. 12, wherein n is A;
  • MIR7A the active chain sequence is set forth in SEQ ID NO. 13, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 14, wherein n is U;
  • MIR8A the active chain sequence is set forth in SEQ ID NO. 15, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO. 16, wherein n is U;
  • MIR9C the active chain sequence is set forth in SEQ ID NO. 17, wherein n is C and the complementary strand sequence is set forth in SEQ ID NO. 18, wherein n is G;
  • MIR10C the active strand sequence is set forth in SEQ ID NO. 19, wherein n is C and the complementary strand sequence is set forth in SEQ ID NO. 20, wherein n is G;
  • MIR11A The active strand sequence is set forth in SEQ ID NO. 21, wherein n is A and the complementary strand sequence is set forth in SEQ ID NO. 22, wherein n is U.
  • MIR12U the active strand sequence is set forth in SEQ ID NO. 23, wherein n is U; the complementary strand sequence is set forth in SEQ ID NO.
  • MIR13A The active strand sequence is set forth in SEQ ID NO. 25, wherein n is A; the complementary strand sequence is set forth in SEQ ID NO.
  • RNA molecule provided by the present invention is mutated to any of the 10th to 22nd positions of the 5' end of the miR-34a-5p sequence to n, and the n is A, C, G or U; Its complementary strand can inhibit the levels of PD-L1, B7-H3, B7-H4 protein in tumor cells, inhibit tumor cell growth and/or proliferation, and thus play an anti-tumor effect. Moreover, studies have shown that the microRNAs provided by the present invention have no significant effect on the secretion of cytokines in serum while anti-tumor.
  • test materials used in the present invention are all commercially available products, all of which are commercially available.
  • HCT-116 Human colorectal cancer cells (HCT-116, HCT-8, Caco-2, SW480), SGC-7901 gastric cancer cells, A549 lung cancer cells, PANC-1 pancreatic cancer cells, and Hela cervical cancer cells (ATCC, USA), Escherichia coli TOP10 (Novagen, USA), cell line tested, no mycoplasma contamination.
  • mice (Balb/c athymic nu+/nu+) and C57BL/6 black mice, 4 weeks old, weighing 18-22 g, were ordered from Shanghai Slack Laboratory Animal Co., Ltd.
  • the experimental methods of the present invention include:
  • All cell lines were cultured in RPMI-1640 or DMEM medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 , and saturated humidity. The cells were grown to about 80% and passaged with 0.25% trypsin. Observe the morphology and growth rate of the cells every day, and replace the fresh medium in time.
  • the cells of the exponential growth phase were seeded in a 24-well plate at a rate of 5 x 10 4 cells/well and cultured overnight until the cells were 80% spliced. After 24 hours, dilute a certain concentration of microRNA to 25 ⁇ L of serum and antibiotic-free RPMI1640 medium, mix gently with a pipette; add 1 ⁇ L of liposome 2000 (Invitrogen) suspension to 25 ⁇ L of serum-free Incubate with antibiotics in RPMI1640 medium for 5 min at room temperature; finally, mix the two together, mix well, and let stand for 20 min at room temperature to fully bind the microRNA to the liposome. Finally, this mixture is added to the cell culture wells.
  • the expression of the protein of interest is detected by standard immunoblotting. 25pg protein was loaded, separated by SDS-PAGE (polyacrylamide gel) electrophoresis, and the protein was electroporated to a nitrocellulose membrane. The PD-L1, B7-H3, and B7-H4 protein primary antibodies were incubated, and then incubated with a 1:1000 enzyme-labeled secondary antibody, and the luminescent reagent was used for color detection; GAPDH was used as an internal reference protein.
  • MicroRNAs were transfected in HCT-116 cells. After 48 hours, they were co-cultured with freshly isolated T cells for 16 hours, supernatants were collected, and cytokines were detected by flow cytometry. First, 120 ⁇ L of magnetic beads of 6 cytokines of IL-2, IL-4, IL-6, IL-10, IFN- ⁇ , and TNF- ⁇ were taken into a flow tube, and allowed to stand in the dark for 30 min. Take 650 ⁇ L of assay buffer and dilute with 250 ⁇ L of the dilution.
  • HCT-116 human colorectal cancer cells were mixed with Matrigel 1:1 ice and inoculated subcutaneously into the left temporal part of nude mice. Each mouse was injected with 100 ⁇ L of a mixed solution of about 5 million cells. Tumors were formed 2 weeks after inoculation. After the tumor was grown to 150-200 mm 3 , the experimental animals were randomly divided into 3 groups of 6 rats each; the solvent control group, the negative control group, and the drug-administered group were given normal saline, negative control nucleic acid sequence, and tested microRNA. .
  • the volume of intravenous administration of each nude mouse was set to 50 ⁇ L, and the dose was 2 nmol/mouse/time; once every two days, a total of five doses were administered.
  • the CCK-8 solution was added to the cell culture well, and the culture was continued for 0.5 hours in the incubator, and the absorbance value (A) at 450 nm was measured by a microplate reader, and the inhibition rate was calculated (sample A value - blank A value) / (control A Value - blank A value) x 100%.
  • microRNAs were randomly selected from SEQ ID NO. 1-22 and tested for their activity of inhibiting the growth of HCT-116 colorectal cancer cells. As a result, as shown in Fig. 2, the activity of these microRNAs to inhibit tumor cell growth was significantly higher than that of miR-34a-5p (including SEQ ID NO. 26: 5'-UGGCAGUGUCUUAGCUGGUUGU-3' and SEQ ID NO.
  • MIR2G comprising SEQ ID NO. 1 and SEQ ID NO. 2, where n is A and U, respectively; MIR9C: comprising SEQ ID NO. 17 and SEQ ID NO.
  • HCT-116 colorectal cancer cells HCT-8 colorectal cancer cells, Caco-2 colorectal cancer cells, SW480 colorectal cancer cells, SGC-7901 gastric cancer cells, A549 lung cancer cells, PANC-1 Antitumor activity in tumor cells such as pancreatic cancer cells and Hela cervical cancer cells.
  • Fig. 3 the growth of various tumor cells was significantly inhibited, indicating that the microRNA has a good broad-spectrum antitumor activity.
  • microRNA is administered (MIR9CM: the sequence comprises SEQ ID NO 17 and SEQ ID NO 18, wherein n is C and G, respectively; SEQ ID NO 18 comprises 22 linked 2'-oxy-methyl groups
  • MIR9CM the sequence comprises SEQ ID NO 17 and SEQ ID NO 18, wherein n is C and G, respectively; SEQ ID NO 18 comprises 22 linked 2'-oxy-methyl groups
  • the ribose nucleotide of the group is treated with a hydroxyl group in the 1, 2, 19, 20, 21 and/or 22 phosphate groups at the 5' end, which is substituted by sulfur, and a hydroxyl group on the 3' terminal ribose is linked to cholesterol.
  • Figure 6 The results are shown in Figure 6.
  • MIR9CM significantly inhibited the growth of HCT-116 cell xenografts in nude mice, but did not affect the body weight and food intake of the mice. Moreover, the tumor volume of the MIR9CM treatment group was significantly smaller than that of miR- at the time of drug withdrawal. Group 34a-5p, p ⁇ 0.05.
  • microRNA down-regulated IL-2 and IL- in HCT-116 cell culture medium. 4. Levels of five cytokines such as IL-6, IL-10 and IFN- ⁇ . Then, we co-cultured HCT-116 cells transfected with microRNA with T cells to observe the effect of tumor cells on the secretion of cytokines by T cells after the action of microRNA. As a result, as shown in Fig.
  • MIR9C did not induce T cells to secrete IL-2, IL-4, IL-6, IL-10, IFN- ⁇ , TNF- ⁇ and other cytokines after acting on tumor cells.
  • MIR9CM high dose microRNA
  • microRNAs To investigate the anti-tumor mechanism of microRNAs, we determined the effect of microRNAs on the expression of negative costimulatory molecules such as PD-L1, B7-H3 and B7-H4. As a result, as shown in Fig. 10, both pairs of microRNAs (MIR1A and MIR9C) significantly inhibited the expression of PD-L1, B7-H3 and B7-H4 proteins in HCT-116 cells. In addition, the protein expression levels of PD-L1, B7-H3 and B7-H4 were lower in the tumor-inoculated tumors of nude mice treated with MIR9CM than in the negative control group (Fig. 11). The present invention has found and confirmed for the first time that PD-L1, B7-H3 and B7-H4 are targets of microRNAs (MIR1A and MIR9C).

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Abstract

提供了miR-34a-5p突变体及其在制备抗肿瘤药物中的应用。该突变体为miR-34a-5p序列5'端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U。该突变体及其互补链能够抑制肿瘤细胞中PD-L1、B7-H3、B7-H4蛋白表达水平，抑制肿瘤细胞生长和/或增殖，同时对血清中细胞因子的分泌没有显著影响。

Description

微小RNA及其在制备抗肿瘤药物中的应用
本申请要求于2017年11月24日提交中国专利局、申请号为201711189410.5、发明名称为“微小RNA及其在制备抗肿瘤药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物制药技术领域，尤其涉及微小RNA及其在制备抗肿瘤药物中的应用。
背景技术
我国恶性肿瘤发病率和死亡率逐年上升，越来越受到医学界的重视，亦然成为基础研究和临床研究的重大课题。虽然肿瘤治疗取得了一定的进展，但离生存期的根本提高对生活质量的要求仍有一定的距离，探求更为安全有效的辅助治疗仍需更多努力。目前，肿瘤免疫治疗已成为继手术、化疗和放疗后的一种有效疗法而被广泛用于临床，尤其是基于免疫卡控点的阻断疗法。有效的抗肿瘤免疫治疗依赖于细胞毒性T淋巴细胞(CTLs)的有效活化，而T细胞的有效活化和功能介导依赖于抗原递呈细胞和T细胞表面的配体/受体对提供的协同刺激信号。其中，B7-CD28是迄今为止公认的最基本的协同刺激信号，包括B7-1、B7-2、B7h、PD-L1(B7-H1)、B7-H2、B7-H3、B7-H4等配体与CD28、CTLA-4、ICOS、PD-1、BTLA等受体提供的促进T细胞生长、分化和产生细胞因子的正性信号，以及限制、终止和/或减弱T细胞应答的负性信号。
PD-L1、B7-H3和B7-H4是近年发现的B7家族协同刺激分子的重要成员，均被发现在多种肿瘤组织中异常高表达，包括肠癌、胃癌、食管癌、肺癌、尿路移形细胞癌、肾癌和前列腺癌等；且其高表达与患者预后差显著相关。大量研究亦已证实，通过抑制PD-L1、B7-H3或B7-H4表达可以获得很好的抗肿瘤效果；目前，已经上市的PD-L1单克隆抗体药物有罗氏的Atezolizumab，用于治疗尿路上皮癌(膀胱癌)或靶向治疗和化疗失败的非小细胞肺癌患者，以及阿斯利康公司的Durvalumab用于治疗局 部晚期或转移性尿路上皮癌患者。我们前期研究发现，PD-L1、B7-H3或B7-H4的表达均可被内源性微小RNA抑制。
微小RNA(microRNA)是近年来发现于真核细胞中的一类长约22个核苷酸的内源性非编码小分子RNA，其5’-端第2-9个碱基(seed region)可通过与靶基因的3′-UTR结合，在翻译水平抑制蛋白合成，从而在基因表达中发挥重要的调节作用。迄今为止，人类基因组中已明确的微小RNA约有2588个(miRBase)，调控着至少30％基因的表达，而每一个微小RNA可能参与调控100～200个靶基因的翻译。微小RNA由于其广泛的调控作用参与了细胞的生长、分化、增殖和凋亡等生命过程，影响着几乎所有的信号通路，并参与各种生理病理过程，特别在肿瘤的发生和发展中发挥了极为重要的作用。
大量研究结果显示微小RNA在肿瘤中呈现异常表达，其中有的微小RNA表达升高而有的降低，分别通过抑制抑癌基因表达或上调癌基因表达，发挥着促癌或抑癌的作用。2002年，Clain等发现两个微小RNA基因miR-15和miR-16在慢性淋巴细胞白血病患者中常发生缺失，首次揭示了微小RNA与肿瘤的密切关系。之后，越来越多的微小RNA被发现在肿瘤中异常表达，如低表达的miR-34a-5p、miR-143、miR-145和高表达的miR-21、miR-27a、miR-155等。业已证实，通过在肿瘤细胞中转入低表达微小RNA的模拟物(mimic和agomir等)，或者转入高表达微小RNA的抑制剂(inhibitor和antagomir等)，可以有效抑制肿瘤细胞增殖、侵袭和转移，从而发挥抗肿瘤效果。
在肿瘤治疗领域，进展最快的微小RNA药物是miR-34a-5p mimic的两性霉素脂质体制剂MRX34。由于miR-34a-5p在细胞和动物水平均显示出了很好的抗肿瘤活性和安全性，美国Mirna Therapeutic公司于2013年推动了一项多中心的I期临床试验，用于治疗原发性肝癌、小细胞肺癌、淋巴瘤、黑色素瘤、多发性骨髓瘤或肾细胞癌患者，也成为第一个进入临床试验的微小RNA药物。随后，RG-101(N-乙酰-D-氨基半乳糖修饰的抗miR-122核酸片段)、RG-012(miR-21抑制剂)、RG-125/AZD4076(N-乙酰半乳糖胺修饰的miR-103/107抑制分子)、MRG-201(miR-29mimic) 和MRG-106(antimiR-155锁核苷酸)等微小RNA药物相继进入临床试验。尽管初步临床试验结果显示MRX34的疗效和安全性尚可，但由于其在5名患者中出现细胞因子风暴而中止了临床试验。因此，进一步研究抗肿瘤活性良好，而不产生不良免疫反应的微小RNA仍是目前的研究热点。
发明内容
有鉴于此，本发明要解决的技术问题在于提供微小RNA及其在制备抗肿瘤药物中的应用。本发明提供的微小RNA抗肿瘤效果良好，且不引起细胞因子异常分泌。
本发明提供了一种RNA分子，miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U。
本发明还提供了与上述RNA分子序列完全互补或部分互补的RNA分子。
具体实施例中，所述部分互补形成的粘性末端为2bp。
具体实施例中，所述粘性末端位于互补RNA分子的3’端。
本发明提供的RNA分子中任意一个或多个碱基经修饰获得的RNA分子。
本发明提供的RNA分子在制备抑制PD-L1、B7-H3、B7-H4等蛋白水平的药物中的应用。
本发明提供的RNA分子在制备抑制肿瘤细胞生长和/或增殖的药物中的应用。
本发明提供的所述RNA分子在制备治疗抗肿瘤药物中的应用。
所述肿瘤为星形细胞瘤、间变性大细胞淋巴瘤、急性淋巴细胞白血病、急性骨髓性白血病、血管肉瘤、乳腺癌、B细胞淋巴瘤、膀胱癌、子宫颈癌、头颈癌、慢性淋巴细胞性白血病、慢性骨髓性白血病、结肠癌、直肠癌、子宫内膜癌、神经胶质瘤、胶质母细胞瘤、胃癌、胃泌素瘤、肝母细胞瘤、肝细胞癌、霍奇金淋巴瘤、卡波西肉瘤白血病、肺癌、平滑肌肉瘤、喉鳞状细胞癌、黑色素瘤、粘膜相关淋巴组织B细胞淋巴瘤、髓母细胞瘤、套细胞淋巴瘤、脑膜瘤、骨髓性白血病、多发性骨髓瘤、高危骨髓增 生异常综合征、间皮瘤、神经纤维瘤、非霍奇金淋巴瘤、非小细胞肺癌、卵巢癌、食管癌、口咽癌骨肉瘤、胰腺癌、乳头状癌、前列腺癌、嗜铬细胞瘤、横纹肌肉瘤、头颈鳞状细胞癌、神经鞘瘤、小细胞肺癌、唾液腺肿瘤、散发性乳头状癌、甲状腺癌、睾丸肿瘤或尿路上皮癌。
本发明还提供了一种抗肿瘤的药物，包括活性链和互补链；
所述活性链为RNA分子或经修饰的该RNA分子，该RNA分子是miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U；所述互补链与活性链互补。
本发明提供的RNA分子为miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U；采用该RNA分子及其互补链能够抑制肿瘤细胞中PD-L1、B7-H3、B7-H4等蛋白水平，抑制肿瘤细胞生长和/或增殖，从而起到抗肿瘤的作用。并且，研究表明，本发明提供的微小RNA在抗肿瘤的同时，对血清中细胞因子的分泌均没有显著影响。
附图说明
图1示miR-449a与miR-34a-5p模拟物抑制肿瘤细胞生长的能力检测结果；其中，图1-a示对HCT-116肠癌细胞的抑制效果；图1-b示对HCT-8肠癌细胞的抑制效果、图1-c示对Caco-2肠癌细胞的抑制效果；图1-d示对SW480肠癌细胞的抑制效果；图1-e示对PANC-1胰腺癌细胞的抑制效果；和图1-f示对Hela宫颈癌细胞的抑制效果；*P＜0.05，**P＜0.01，***P＜0.001；
图2示本发明的实施例中，微小RNA(MIR1A、MIR2G、MIR3A、MIR4C、MIR5U、MIR6U、MIR7A、MIR8A、MIR9C、MIR10C、MIR11A、MIR12U和MIR13A)模拟物与miR-34a-5p处理的HCT-116结直肠癌细胞的相对生长能力检测结果；*P＜0.05，**P＜0.01，***P＜0.001；
图3示本发明的实施例中，微小RNA(MIR1A和MIR9C)和miR-34a-5p模拟物抑制肿瘤细胞生长的能力检测结果；其中，图3-a示对HCT-116肠癌细胞的抑制效果；图3-b示对HCT-8肠癌细胞的抑制效果、图3-c示对Caco-2肠癌细胞的抑制效果；图3-d示对SW480肠癌细胞的 抑制效果；图3-e示对SGC-7901胃癌细胞的抑制效果；图3-f示对A549肺癌细胞的抑制效果；图3-g示对PANC-1胰腺癌细胞的抑制效果；和图3-h示对Hela宫颈癌细胞的抑制效果；
图4示本发明的实施例中，不同浓度(0.1、2.0、10、25、50、100nM)的微小RNA(MIR1A和MIR9C)和miR-34a-5p模拟物抑制HCT-116肿瘤细胞生长的能力检测结果；其中，图4-a示miR-34a-5p的抑制效果；图4-b示MIR1A的抑制效果、图4-c示MIR9C的抑制效果；
图5示本发明的实施例中，平端或粘性末端的微小RNA(MIR1A和MIR9C)和miR-34a-5p模拟物抑制HCT-116肿瘤细胞生长的能力检测结果；其中，图5-a示平端miR-34a-5p与粘性末端miR-34a-5p的抑制效果；图5-b示平端MIR1A与粘性末端MIR1A的抑制效果、图5-c示平端MIR9C与粘性末端MIR9C的抑制效果；
图6示本发明的实施例中，微小RNA(MIR9CM)和miR-34a-5p激动剂抑制HCT-116细胞裸鼠移植瘤生长的能力检测结果；其中，图6-a示移植瘤的生长曲线；图6-b示荷瘤裸鼠体重变化曲线；图6-c示荷瘤裸鼠摄食量变化曲线；
图7示本发明的实施例中，微小RNA(MIR9C)模拟物处理的HCT-116细胞中细胞因子分泌水平的流式细胞术检测结果；其中，图7-a示IL-2蛋白的检测结果；图7-b示IL-4蛋白的检测结果；图7-c示IL-6蛋白的检测结果；图7-d示IL-10蛋白的检测结果；图7-e示TNF-α蛋白的检测结果；图7-f示IFN-γ蛋白的检测结果；
图8示本发明的实施例中，微小RNA(MIR9C)模拟物处理的HCT-116细胞/T淋巴细胞共培养体系中细胞因子分泌水平的流式细胞术检测结果；其中，图8-a示IL-2蛋白的检测结果；图8-b示IL-4蛋白的检测结果；图8-c示IL-6蛋白的检测结果；图8-d示IL-10蛋白的检测结果；图8-e示TNF-α蛋白的检测结果；图8-f示IFN-γ蛋白的检测结果；
图9示本发明的实施例中，微小RNA(MIR9CM)激动剂处理的C57BL/6小黑鼠血清中细胞因子分泌水平的流式细胞术检测结果；其中，图9-a示IL-2蛋白的检测结果；图9-b示IL-4蛋白的检测结果；图9-c 示IL-6蛋白的检测结果；图9-d示IL-10蛋白的检测结果；图9-e示TNF-α蛋白的检测结果；图9-f示IFN-γ蛋白的检测结果；
图10示本发明的实施例中，微小RNA(MIR1A和MIR9C)和miR-34a-5p模拟物处理的HCT-116细胞中PD-L1、B7-H3和B7-H4蛋白的Western bolt检测结果；
图11示本发明的实施例中，微小RNA(MIR9CM)激动剂处理的HCT-116细胞移植瘤中蛋白表达的Western bolt检测结果；其中，图11-a示PD-L1蛋白的检测结果；图11-b示B7-H3蛋白的检测结果；图11-c示B7-H4蛋白的检测结果。
具体实施方式
本发明提供了微小RNA及其在制备抗肿瘤药物中的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明说明书中提及的术语定义如下：
本发明提供的RNA分子为微小RNA(microRNA，或miRNA)，其是指单链的寡核糖核酸。核糖核苷酸是由核苷酸经磷酸二酯键缩合而成长链状分子。核糖核苷酸分子由一分子碱基、一分子核糖和磷酸构成。本发明提供的miRNA的碱基有4种，A腺嘌呤、G鸟嘌呤、C胞嘧啶、U尿嘧啶，5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、6-甲基和腺嘌呤和鸟嘌呤的其它烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基和其它烷基衍生物、5-硫尿嘧啶和胞嘧啶、5-丙炔基尿嘧啶和嘧啶碱基的其他炔基衍生物、6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤代、8-氨基、8-巯基、8-硫代烷基、8-羟基和其他8-取代的腺嘌呤和鸟嘌呤、5-卤素(包括5-溴、5-三氟甲基和其它5-取代的尿嘧啶和胞嘧啶)、7-甲基鸟嘌呤和7-甲基腺嘌呤、2-F-腺嘌呤、2-氨基腺嘌呤、8-氮鸟嘌呤和8-氮杂腺嘌呤、7-脱氮鸟嘌呤和7- 脱氮腺嘌呤、3-脱氮鸟嘌呤和3-脱氮腺嘌呤。
所述碱基，可以是无修饰碱基，也可以是修饰碱基。
所述修饰碱基，是指碱基连接基团包括但不限于NH 2、生物素、胺基、低级胺基烷基、低级烷基、NHCOCH 3、乙酰基、2′-氧基-甲基(2′O-Me)、DMTO、荧光素、硫醇或吖啶。
所述修饰碱基，是指本发明提供的RNA分子链或其互补链中的1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个碱基连接修饰基团。
所述修饰，是指本发明提供的RNA分子链或其互补链中的碱基连接任何一种或多种基团或其组合。
所述核糖，可以是无修饰核糖，也可以是修饰核糖。
所述修饰核糖，是指核糖连接基团包括但不限于低级烷基、烯基、炔基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH 3、Cl、Br、CN、OCN、CF 3、OCF 3、SOCH 3、SO 2CH 3、ONO 2、NO 2、N 3、NH 2、杂环烷基、杂环氨基烷基、氨基烷基、聚氨基烷基、取代的甲硅烷基、RNA裂解基团、嵌入剂、用于改善微小RNA的药代动力学性质的基团或用于改善微小RNA的药效学性质的基团，以及具有相似性质的其它取代基。另外的糖取代基包括2′-O-2-甲氧基乙基(2′-O-CH 2CH 2OCH 3)、2′-二甲基氨氧基乙氧基[2′-O-CH 2-O-CH 2-N(CH 3) 2]、烯丙基(-CH 2-CH＝CH 2)、-O-烯丙基(-O-CH 2-CH-CH 2)、甲氧基(-O-CH 3)、氨基丙氧基(-OCH 2CH 2CH 2NH 2)和氟(F)等。
所述修饰核糖，是指本发明提供的RNA分子链或其互补链中的任何1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21或22个核糖连接修饰基团。
所述修饰，是指本发明提供的RNA分子链或其互补链中的核糖连接任何一种或多种基团或其组合。
所述互补，又称互补配对，是指碱基通过氢键与它互补的碱基相连。互补碱基对A于U之间形成两对氢键，C与G之间形成三对氢键。
所述完全互补是指序列完全匹配且不形成粘性末端。所述部分互补是 指序列完全匹配，但形成粘性末端。
本发明所述突变是指一个碱基被另一个碱基所取代，突变后，RNA分子中碱基的数量不发生改变。本发明中，所述突变不存在碱基被与自身相同碱基取代，即所述突变不存在A被A取代，C被C取代，G被G取代，U被U取代的情况。
本发明提供的RNA分子为miR-34a序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U。
本发明还提供了与上述RNA分子序列完全互补或部分互补的RNA分子。
具体实施例中，所述部分互补形成的粘性末端为2bp。
具体实施例中，所述粘性末端位于互补RNA分子的3’端。
本发明中，提供的突变的miR-34a-5p记为活性链，与其完全互补或部分互补的RNA分子记为互补链。
所述miR-34a-5p序列如SEQ ID NO.26所示。
一些实施例中，所述完全互补的微小RNA分子对包括SEQ ID NO：1所示序列的活性链和SEQ ID NO.27所示序列的互补链；或者包括SEQ ID NO：17所示序列的活性链和SEQ ID NO.28所示序列的互补链。
部分互补的微小RNA分子对包括：
活性链序列如SEQ ID NO.1所示，其互补链序列如SEQ ID NO.2所示。
SEQ ID NO.1为miR-34a-5p序列中5’端第10位碱基C突变为n，所述n为A、G或U。具体实施例中，SEQ ID NO.1中所述n为A。SEQ ID NO.1中5’端第1～20位与SEQ ID NO：2中5’端第1～20位互补；SEQ ID NO.1的5’端第21～22位形成粘性末端；SEQ ID NO.2的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.3所示，其互补链序列如SEQ ID NO.4所示。
SEQ ID NO.3为miR-34a-5p序列中5’端第11位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.3中所述n为G。SEQ ID  NO.3中5’端第1～20位与SEQ ID NO：4中5’端第1～20位互补；SEQ ID NO.3的5’端第21～22位形成粘性末端；SEQ ID NO.4的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.5所示，其互补链序列如SEQ ID NO.6所示。
SEQ ID NO.5为miR-34a-5p序列中5’端第12位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.5中所述n为A。SEQ ID NO.5中5’端第1～20位与SEQ ID NO：6中5’端第1～20位互补；SEQ ID NO.5的5’端第21～22位形成粘性末端；SEQ ID NO.6的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.7所示，其互补链序列如SEQ ID NO.8所示。
SEQ ID NO.7为miR-34a-5p序列中5’端第13位碱基A突变为n，所述n为C、G或U。具体实施例中，SEQ ID NO.7中所述n为C。SEQ ID NO.7中5’端第1～20位与SEQ ID NO：8中5’端第1～20位互补；SEQ ID NO.7的5’端第21～22位形成粘性末端；SEQ ID NO.8的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.9所示，其互补链序列如SEQ ID NO.10所示。
SEQ ID NO.9为miR-34a-5p序列中5’端第14位碱基G突变为n，所述n为A、C或U。具体实施例中，SEQ ID NO.9中所述n为U。SEQ ID NO.9中5’端第1～20位与SEQ ID NO：10中5’端第1～20位互补；SEQ ID NO.9的5’端第21～22位形成粘性末端；SEQ ID NO.10的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.11所示，其互补链序列如SEQ ID NO.12所示。
SEQ ID NO.11为miR-34a-5p序列中5’端第15位碱基C突变为n，所述n为A、G或U。具体实施例中，SEQ ID NO.11中所述n为U。SEQ ID NO.11中5’端第1～20位与SEQ ID NO：12中5’端第1～20位互补；SEQ  ID NO.11的5’端第21～22位形成粘性末端；SEQ ID NO.12的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.13所示，其互补链序列如SEQ ID NO.14所示。
SEQ ID NO.13为miR-34a-Sp序列中5’端第16位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.13中所述n为A。SEQ ID NO.13中5’端第1～20位与SEQ ID NO：14中5’端第1～20位互补；SEQ ID NO.13的5’端第21～22位形成粘性末端；SEQ ID NO.14的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.15所示，其互补链序列如SEQ ID NO.16所示。
SEQ ID NO.15为miR-34a-5p序列中5’端第17位碱基G突变为n，所述n为A、C或U。具体实施例中，SEQ ID NO.15中所述n为A。SEQ ID NO.15中5’端第1～20位与SEQ ID NO：16中5’端第1～20位互补；SEQ ID NO.15的5’端第21～22位形成粘性末端；SEQ ID NO.16的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.17所示，其互补链序列如SEQ ID NO.18所示。
SEQ ID NO.17为miR-34a-5p序列中5’端第18位碱基G突变为n，所述n为A、C或U。具体实施例中，SEQ ID NO.17中所述n为C。SEQ ID NO.17中5’端第1～20位与SEQ ID NO：18中5’端第1～20位互补；SEQ ID NO.17的5’端第21～22位形成粘性末端；SEQ ID NO.18的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.19所示，其互补链序列如SEQ ID NO.20所示。
SEQ ID NO.19为miR-34a-5p序列中5’端第19位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.19中所述n为C。SEQ ID NO.19中5’端第1～20位与SEQ ID NO：20中5’端第1～20位互补；SEQ ID NO.19的5’端第21～22位形成粘性末端；SEQ ID NO.20的5’端第21～22 位形成粘性末端。
活性链序列如SEQ ID NO.21所示，其互补链序列如SEQ ID NO.22所示。
SEQ ID NO.21为miR-34a-5p序列中5’端第20位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.21中所述n为A。SEQ ID NO.21中5’端第1～20位与SEQ ID NO：22中5’端第1～20位互补；SEQ ID NO.21的5’端第21～22位形成粘性末端；SEQ ID NO.22的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.23所示，其互补链序列如SEQ ID NO.24所示。
SEQ ID NO.23为miR-34a-5p序列中5’端第21位碱基G突变为n，所述n为A、C或U。具体实施例中，SEQ ID NO.23中所述n为U。SEQ ID NO.23中5’端第1～20位与SEQ ID NO：24中5’端第1～20位互补；SEQ ID NO.23的5’端第21～22位形成粘性末端；SEQ ID NO.24的5’端第21～22位形成粘性末端。
活性链序列如SEQ ID NO.25所示，其互补链序列如SEQ ID NO.24所示。
SEQ ID NO.25为miR-34a-5p序列中5’端第22位碱基U突变为n，所述n为A、C或G。具体实施例中，SEQ ID NO.25中所述n为A。SEQ ID NO.25中5’端第1～20位与SEQ ID NO：24中5’端第1～20位互补；SEQ ID NO.25的5’端第21～22位形成粘性末端；SEQ ID NO.24的5’端第21～22位形成粘性末端。
本发明提供的RNA分子中任意一个或多个碱基经修饰获得的RNA分子。
本发明实施例中，对互补链进行修饰。所述修饰包括：在序列中的任意一个或多个核糖中连接2′-氧基-甲基基团、使任意一个或多个磷酸基团中的羟基被硫取代、使3’末端的核糖上的羟基连接胆固醇。
本发明提供的RNA分子在制备抑制肿瘤细胞生长和/或增殖的药物中的应用。
通过实验验证，本发明提供的微小RNA分子对对各种肿瘤细胞的生长均有显著的抑制作用，显示本发明提供的微小RNA分子对具有很好的广谱抗肿瘤活性；且结果表明，本发明提供的微小RNA分子对对肿瘤细胞的抑制作用优于miR-34a-5p。
本发明进行实验的肿瘤细胞包括结直肠癌细胞、胃癌细胞、肺癌细胞、胰腺癌细胞和/或宫颈癌细胞。
具体的，进行实验的肿瘤细胞为HCT-116结直肠癌细胞、HCT-8结直肠癌细胞、Caco-2结直肠癌细胞、SW480结直肠癌细胞、SGC-7901胃癌细胞、A549肺癌细胞、PANC-1胰腺癌细胞和Hela宫颈癌细胞等肿瘤细胞中的抗肿瘤活性。
进行实验的微小RNA分子对中，一些对肿瘤细胞生长的抑制活性与miR-34a-5p相当，这些微小RNA分子对中的活性链的序列如下任一种所示：SEQ ID NO.1、SEQ ID NO.11、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.21。
具体的，这些分子对为：
MIR1A：活性链序列如SEQ ID NO.1所示，其中n为A；其互补链序列如SEQ ID NO.2所示，其中n为U；
MIR6U：活性链序列如SEQ ID NO.11所示，其中n为U，其互补链序列如SEQ ID NO.12所示，其中n为A；
MIR9C：活性链序列如SEQ ID NO.17所示，其中n为C，其互补链序列如SEQ ID NO.18所示，其中n为G；
MIR10C：活性链序列如SEQ ID NO.19所示，其中n为C，其互补链序列如SEQ ID NO.20所示，其中n为G；
MIR11A：活性链序列如SEQ ID NO.21所示，其中n为A，其互补链序列如SEQ ID NO.22所示，其中n为U。
上述微小RNA分子对，对肿瘤细胞生长的抑制活性与miR-34a-5p相当，但它们相对于miR-34a-5p而言，具有更高的用药安全性。实验结果显示，其中MIR9C对实验小鼠血清中IL-4、IL-6、IL-10、IFN-γ、TNF-α等5种细胞因子的分泌均没有显著影响。
进行实验的微小RNA分子对中，一些对肿瘤细胞生长的抑制活性显著优于miR-34a-5p，p＜O.05，这些微小RNA分子对中的活性链的序列如下任一种所示：SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.23、SEQ ID NO.25。
具体的，这些分子对为：
MIR2G：活性链序列如SEQ ID NO.3所示，其中n为G；其互补链序列如SEQ ID NO.4所示，其中n为C；
MIR3A：活性链序列如SEQ ID NO.5所示，其中n为A；其互补链序列如SEQ ID NO.6所示，其中n为U；
MIR4C：活性链序列如SEQ ID NO.7所示，其中n为C；其互补链序列如SEQ ID NO.8所示，其中n为G；
MIR5U：活性链序列如SEQ ID NO.9所示，其中n为U；其互补链序列如SEQ ID NO.10所示，其中n为A；
MIR7A：活性链序列如SEQ ID NO.13所示，其中n为A；其互补链序列如SEQ ID NO.14所示，其中n为U；
MIR8A：活性链序列如SEQ ID NO.15所示，其中n为A；其互补链序列如SEQ ID NO.16所示，其中n为U；
MIR12U：活性链序列如SEQ ID NO.23所示，其中n为U；其互补链序列如SEQ ID NO.24所示；
MIR13A：活性链序列如SEQ ID NO.25所示，其中n为A；其互补链序列如SEQ ID NO.24所示。
本发明提供的RNA分子在制备抑制PD-L1、B7-H3、B7-H4等蛋白水平的药物中的应用。
本发明测定了微小RNA分子对，对PD-L1、B7-H3和B7-H4等负性协同刺激分子表达的影响。通过实验验证，本发明提供的微小RNA分子对显著抑制了HCT-116细胞中PD-L1、B7-H3和B7-H4蛋白表达。
用于测定负性协同刺激分子表达的微小RNA分子对中的活性链序列为SEQ ID NO.1或SEQ ID NO.17。
具体的，这些分子对为：
MIR1A：活性链序列如SEQ ID NO.1所示，其中n为A；其互补链序列如SEQ ID NO.2所示，其中n为U；
MIR9C：活性链序列如SEQ ID NO.17所示，其中n为C，其互补链序列如SEQ ID NO.18所示，其中n为G；
在裸鼠中接种HCT-116细胞构建肿瘤模型。待肿瘤长至150-200mm 3后，给予本发明提供的微小RNA进行治疗。结果表明，微小RNA显著抑制了裸鼠中HCT-116细胞移植瘤的生长，但不影响小鼠的体重和摄食量；而且，在刚停药时本发明提供微小RNA治疗组肿瘤体积明显小于miR-34a-5p组。
本发明提供的所述RNA分子在制备治疗抗肿瘤药物中的应用。
本发明进行抗肿瘤实验的RNA分子中，活性链的序列如SEQ ID NO.17所示，其中n为C；互补链的序列如SEQ ID NO.18所示，其中n为G。
进行抗肿瘤实验的RNA分子中，互补链经过修饰。所述修饰为，SEQ ID NO.18中每个核糖分子皆连接2′-氧基-甲基基团，其5’端第1、2、19、20、21和/或22个磷酸基团中的羟基被硫取代，3’末端核糖上的羟基连接胆固醇。
所述肿瘤为星形细胞瘤、间变性大细胞淋巴瘤、急性淋巴细胞白血病、急性骨髓性白血病、血管肉瘤、乳腺癌、B细胞淋巴瘤、膀胱癌、子宫颈癌、头颈癌、慢性淋巴细胞性白血病、慢性骨髓性白血病、结肠癌、直肠癌、子宫内膜癌、神经胶质瘤、胶质母细胞瘤、胃癌、胃泌素瘤、肝母细胞瘤、肝细胞癌、霍奇金淋巴瘤、卡波西肉瘤白血病、肺癌、平滑肌肉瘤、喉鳞状细胞癌、黑色素瘤、粘膜相关淋巴组织B细胞淋巴瘤、髓母细胞瘤、套细胞淋巴瘤、脑膜瘤、骨髓性白血病、多发性骨髓瘤、高危骨髓增生异常综合征、间皮瘤、神经纤维瘤、非霍奇金淋巴瘤、非小细胞肺癌、卵巢癌、食管癌、口咽癌骨肉瘤、胰腺癌、乳头状癌、前列腺癌、嗜铬细胞瘤、横纹肌肉瘤、头颈鳞状细胞癌、神经鞘瘤、小细胞肺癌、唾液腺肿瘤、散发性乳头状癌、甲状腺癌、睾丸肿瘤或尿路上皮癌。
本发明进一步验证了完全互补的微小RNA分子对及部分互补的微小 RNA分子对的抗肿瘤活性。结果表明，所有粘性末端微小RNA的抗肿瘤活性均显著强于与之对应的平端微小RNA，表明粘性末端微小RNA具有更强的抗肿瘤活性。
本发明还提供了一种抗肿瘤的药物，包括活性链和互补链；
所述活性链为RNA分子或经修饰的该RNA分子，该RNA分子是miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U；所述互补链与活性链互补。
本发明实施例中，所述抗肿瘤药物中，包含的活性链序列为：SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.15、SEQ ID NO.17、SEQ ID NO.19、SEQ ID NO.21、SEQ ID NO.23、SEQ ID NO.25。
具体的，包括如下微小RNA分子对：
MIR1A：活性链序列如SEQ ID NO.1所示，其中n为A；其互补链序列如SEQ ID NO.2所示，其中n为U；
MIR2G：活性链序列如SEQ ID NO.3所示，其中n为G；其互补链序列如SEQ ID NO.4所示，其中n为C；
MIR3A：活性链序列如SEQ ID NO.5所示，其中n为A；其互补链序列如SEQ ID NO.6所示，其中n为U；
MIR4C：活性链序列如SEQ ID NO.7所示，其中n为C；其互补链序列如SEQ ID NO.8所示，其中n为G；
MIR5U：活性链序列如SEQ ID NO.9所示，其中n为U；其互补链序列如SEQ ID NO.10所示，其中n为A；
MIR6U：活性链序列如SEQ ID NO.11所示，其中n为U，其互补链序列如SEQ ID NO.12所示，其中n为A；
MIR7A：活性链序列如SEQ ID NO.13所示，其中n为A；其互补链序列如SEQ ID NO.14所示，其中n为U；
MIR8A：活性链序列如SEQ ID NO.15所示，其中n为A；其互补链序列如SEQ ID NO.16所示，其中n为U；
MIR9C：活性链序列如SEQ ID NO.17所示，其中n为C，其互补链 序列如SEQ ID NO.18所示，其中n为G；
MIR10C：活性链序列如SEQ ID NO.19所示，其中n为C，其互补链序列如SEQ ID NO.20所示，其中n为G；
MIR11A：活性链序列如SEQ ID NO.21所示，其中n为A，其互补链序列如SEQ ID NO.22所示，其中n为U。
MIR12U：活性链序列如SEQ ID NO.23所示，其中n为U；其互补链序列如SEQ ID NO.24所示；
MIR13A：活性链序列如SEQ ID NO.25所示，其中n为A；其互补链序列如SEQ ID NO.24所示。
本发明提供的RNA分子为miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U；采用该RNA分子及其互补链能够抑制肿瘤细胞中PD-L1、B7-H3、B7-H4蛋白水平，抑制肿瘤细胞生长和/或增殖，从而起到抗肿瘤的作用。并且，研究表明，本发明提供的微小RNA在抗肿瘤的同时，对血清中细胞因子的分泌均没有显著影响。
本发明采用的试材皆为普通市售品，皆可于市场购得。
10×Taq Bufer with KCl、MgCl 2、dNTPs、Taq DNA聚合酶、GeneRuler 100bp DNA Ladder(Thermo，美国)，西班牙琼脂糖(SunShineBio)，6×DNA Loading Dye、反转录试剂盒(TaKaRa，日本)，PCR引物(苏州金唯智生物科技有限公司)，GelRed Nucleic Acid Gel Stain(Biotium，美国)，Triton-100、MgCl 2·6H 2O、EDTA、NaCl、三(羟甲基)氨基甲烷(Tris)、十二烷基磺酸钠(SDS)、吐温-20、二甲亚砜、甘氨酸、盐酸、蔗糖、苯酚、氯仿、异戊醇、无水乙醇(国药集团化学试剂有限公司)，胎牛血清、opti-MEM(Gibco，美国)，脂质体2000、Trizol(Invitrogen，美国)，DMEM培养基、胰蛋白酶(Hyclone，美国)，切胶回收试剂盒和质粒抽提试剂盒(Axygen，美国)，BCA蛋白定量试剂盒；氨卞青霉素(北京索莱宝科技有限公司)，酵母提取物、蛋白胨(Oxoid，英国)；CCK8(东仁化学科技(上海)有限公司，日本)，蛋白酶抑制剂(Roche，瑞士)，Protein G sepharose beads(GE，美国)，RIPA裂解液、SDS-PAGE蛋白上样缓冲液 (碧云天生物技术研究所，中国)，Protein Ladder和二抗(Santa Cruz，美国)，PD-L1、B7-H3、B7-H4蛋白一抗(Santa Cruz，美国)；ECL化学发光试剂盒、NC膜、PVDF膜(密理博，德国)，去离子水(由上海和泰Master-S超纯水机制备)，微小RNA(上海吉玛基因有限公司化学合成；HPLC纯化，纯度＞97％)；Matrigel(BD，美国)；流式细胞因子检测试剂盒BD cytometric bead array(CBA)human th1/th2/th17 cytokine kit(BD，美国)。
枪尖、1.5mLEP管(Axygen，美国)，细胞培养皿及培养板、离心管(Coming，美国)，电子天平(Scount SE，中国)，微波炉(Media，中国)；PCR仪、水平电泳仪、垂直电泳仪、转膜仪、凝胶成像系统(Bio-Rad，美国)；流式细胞仪(Beckman，美国)；移液枪、高速离心机(Eppendorf，德国)，紫外分光光度计(Q5000，美国)，平行震荡仪、高速低温离心机、细胞培养箱、-80℃超低温冰箱(Thermo Scientific，美国)，常速离心机(Joμan，法国)，倒置显微镜及荧光显微镜(Olympμs，日本)，微量紫外可见光分光光度计(Q5000，美国)，电热恒温培养箱(PYX-DHS，中国)，电热恒温水浴箱(HH-S型，中国)，恒温振荡培养箱(HZQ-X100，中国)，酶标仪(Tecan，瑞士)，游标卡尺，1ml注射器(江苏正康医疗器械有限公司，中国)。
人结直肠癌细胞(HCT-116、HCT-8、Caco-2、SW480)、SGC-7901胃癌细胞、A549肺癌细胞、PANC-1胰腺癌细胞和Hela宫颈癌细胞(ATCC，美国)，大肠杆菌TOP10(Novagen，美国)，细胞株经检测，无支原体污染。
裸鼠(Balb/c athymic nu+/nu+)和C57BL/6小黑鼠，4周龄，体重18～22g，订购于上海斯莱克实验动物有限公司。
下面结合实施例，进一步阐述本发明：
进行实验的RNA序列如表1：
表1 微小RNA序列
Figure PCTCN2018077481-appb-000001
Figure PCTCN2018077481-appb-000002
本发明的实验方法包括：
1、细胞培养
所有细胞株均采用含10％胎牛血清的RPMI-1640或DMEM培养基进行培养，培养条件为37℃、5％CO 2、饱和湿度。细胞生长至80％左右，用0.25％胰酶消化传代。每天观察细胞的形态及生长速度，及时更换新鲜培养基。
2、转染微小RNA
按5×10 4个细胞/孔的量在24孔板中接种指数生长期的细胞，培养过夜，直到细胞80％汇片。24小时后，将一定浓度的微小RNA稀释至25μL不含血清和抗生素的RPMI1640培养基中，用移液枪轻轻混合均匀；将1μL脂质体2000(Invitrogen公司)悬液加入25μL不含血清和抗生素的RPMI1640培养基中，室温孵育5min；最后将两者混合在一起，充分混匀，室温静置20min，使微小RNA与脂质体充分结合。最后，将此混合物加入细胞培养孔中。
3、蛋白免疫印迹检测
标准免疫印迹方法检测目的蛋白表达。25pg蛋白上样，SDS-PAGE(聚丙烯酰胺凝胶)电泳分离，蛋白电转至硝酸纤维素膜。PD-L1、B7-H3、B7-H4蛋白一抗孵育，然后用1∶1000酶标二抗孵育，发光试剂显色检测；GAPDH作为内参蛋白。
4、流式细胞术检测细胞因子
在HCT-116细胞中转染微小RNA。48小时后，将其与新分离T细胞共培养16小时，收集上清，流式细胞仪检测细胞因子。首先，各取IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α共6种细胞因子的磁珠120μL至流式管中，避光静置30min。取650μL检测缓冲液，用250μL稀释液稀释。取出5只流式管，分别加入40μL稀释后的检测缓冲液、40μL混合均匀的磁珠、40μL按照1∶256、1∶128、1∶64、1∶32和1∶16稀释的标准品，制作标准曲线。另取流式管，分别加入40μL稀释后的检测缓冲液、40μL混合均匀的磁珠和40μL样品。避光静置2h，每隔30min震荡一次。每管依次加入1mL清洗缓冲液，200g离心5min，弃上清。再加入300μL鞘液与底部磁珠形成混悬液，流式仪检测。
5、动物实验
HCT-116人结直肠癌细胞与基质胶1∶1冰上混合，接种于裸鼠左侧腋部皮下。每只鼠注射100μL混合液，约500万细胞。接种后2周成瘤。待肿瘤长至150-200mm 3后，将实验动物随机分为3组，每组6只鼠；溶剂对照组、阴性对照组、给药组分别给予生理盐水、阴性对照核酸序列、受试微小RNA。每只裸鼠静脉注射给药体积设置为50μL，剂量为2nmol/ 鼠/次；每两天注射一次，一共给药五次。从给药第一天开始，每3天测定肿瘤大小和裸鼠体重一次；肿瘤大小使用公式V＝(a×b 2)/2计算，其中a和b分别是肿瘤的长径和短径。
6、CCK8法检测细胞活性
向细胞培养孔中加入CCK-8溶液，在培养箱中继续培养0.5小时后，酶标仪检测450nm处吸光值(A)，计算抑制率＝(样品A值-空白A值)/(对照A值-空白A值)×100％。
本发明的实验结果为：
从SEQ ID NO.1-22中随机选择13条微小RNA，测定其抑制HCT-116结直肠癌细胞生长的活性。结果如图2所示，这些微小RNA抑制肿瘤细胞生长的活性均显著高于miR-34a-5p(包含SEQ ID NO.26：5’-UGGCAGUGUCUUAGCUGGUUGU-3’和SEQ ID NO.24：5’-AACCAGCUAAGACACUGCCAUU-3’)，如MIR2G、MIR3A、MIR4C、MIR5U、MIR7A、MIR8A、MIR12U和MIR13A等，或与之相当(如MIR1A、MIR6U、MIR9C、MIR10C和MIR11A等)。再选取两对微小RNA(MIR1A：包含SEQ ID NO.1和SEQ ID NO.2，其中n分别为A和U；MIR9C：包含SEQ ID NO.17和SEQ ID NO.18，其中n分别为C和G)进一步研究其在HCT-116结直肠癌细胞、HCT-8结直肠癌细胞、Caco-2结直肠癌细胞、SW480结直肠癌细胞、SGC-7901胃癌细胞、A549肺癌细胞、PANC-1胰腺癌细胞和Hela宫颈癌细胞等肿瘤细胞中的抗肿瘤活性。结果如图3所示，各种肿瘤细胞的生长均被显著抑制，显示微小RNA具有很好的广谱抗肿瘤活性。
此外，我们还分别比较了活性链序列完全相同的粘性末端MIR1A和MIR9C与平端MIR1A(包含SEQ ID NO.1和SEQ ID NO.27：5’-ACAACCAGCUAAUACACUGCCA-3’)和平端MIR9C(包含SEQ ID NO.17和SEQ ID NO.28：5’-ACAAGCAGCUAAGACACUGCCA-3’)抑制HCT-116细胞生长的作用，同时也比较了粘性末端miR-34a-5p(包含SEQ ID NO.26和SEQ ID NO.24)与平端miR-34a-5p(包含SEQ ID NO.26和SEQ ID NO.29：5’-ACAACCAGCUAAGACACUGCCA-3’)的活性差 异。结果如图5所示，在这三对微小RNA中，所有粘性末端微小RNA的抗肿瘤活性均显著强于与之对应的平端微小RNA，表明粘性末端微小RNA具有更强的抗肿瘤活性。
我们在裸鼠中接种HCT-116细胞构建肿瘤模型。待肿瘤长至150-200mm 3后，给予微小RNA(MIR9CM：序列包含SEQ ID NO17和SEQ ID NO18，其中n分别为C和G；SEQ ID NO18包含22个连接2′-氧基-甲基基团的核糖的核苷酸，其5’端第1、2、19、20、21和/或22个磷酸基团中的羟基被硫取代，3’末端核糖上的羟基连接胆固醇)进行治疗。结果如图6所示，MIR9CM显著抑制了裸鼠中HCT-116细胞移植瘤的生长，但不影响小鼠的体重和摄食量；而且，在刚停药时MIR9CM治疗组肿瘤体积显著小于miR-34a-5p组，p＜0.05。
我们采用体内外实验研究了微小RNA的安全性。首先，我们将微小RNA直接作用于肿瘤细胞，观察其对肿瘤细胞分泌细胞因子的影响；结果如图7所示，微小RNA(MIR9C)下调了HCT-116细胞培养液中IL-2、IL-4、IL-6、IL-10、IFN-γ等5种细胞因子的水平。然后，我们将转染有微小RNA的HCT-116细胞与T细胞共培养，观察微小RNA作用后肿瘤细胞诱导T细胞分泌细胞因子的影响。结果如图8所示，MIR9C作用于肿瘤细胞后不会诱导T细胞分泌IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α等细胞因子。最后，在小鼠中静脉注射高剂量微小RNA(MIR9CM)后，测定小鼠血清中细胞因子水平；结果如图9所示，除IL-2外，MIR9CM对小鼠血清中IL-4、IL-6、IL-10、IFN-γ、TNF-α等5种细胞因子的分泌均没有显著影响。
为了研究微小RNA的抗肿瘤机制，我们测定了微小RNA对PD-L1、B7-H3和B7-H4等负性协同刺激分子表达的影响。结果如图10所示，两对微小RNA(MIR1A和MIR9C)均显著抑制了HCT-116细胞中PD-L1、B7-H3和B7-H4蛋白表达。此外，在经MIR9CM治疗后的裸鼠接种肿瘤中，PD-L1、B7-H3和B7-H4的蛋白表达水平均低于阴性对照组(图11)。本发明首次发现并证实PD-L1、B7-H3和B7-H4是微小RNA(MIR1A和MIR9C)的作用靶点。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2018077481-appb-000003
Figure PCTCN2018077481-appb-000004
Figure PCTCN2018077481-appb-000005
Figure PCTCN2018077481-appb-000006
Figure PCTCN2018077481-appb-000007
Figure PCTCN2018077481-appb-000008
Figure PCTCN2018077481-appb-000009
Figure PCTCN2018077481-appb-000010
Figure PCTCN2018077481-appb-000011
Figure PCTCN2018077481-appb-000012
Figure PCTCN2018077481-appb-000013

Claims (10)

  1. 一种RNA分子，其特征在于，miR-34a-5p序列5’端第10位～第22位中的任一碱基突变为n，所述n为A、C、G或U。
  2. 与权利要求1所述RNA分子序列完全互补或部分互补的RNA分子。
  3. 根据权利要求2所述的RNA分子，其特征在于，所述部分互补形成的粘性末端为2bp。
  4. 根据权利要求3所述的RNA分子，所述粘性末端位于权利要求2所述RNA分子的3’端。
  5. 权利要求1～4任一项所述RNA分子中任意一个或多个碱基被修饰的RNA分子。
  6. 权利要求1～5任一项所述RNA分子在制备抑制PD-L1、B7-H3、B7-H4蛋白水平的药物中的应用。
  7. 权利要求1～5任一项所述RNA分子在制备抑制肿瘤细胞生长和/或增殖的药物中的应用。
  8. 权利要求1～5任一项所述RNA分子在制备治疗抗肿瘤药物中的应用。
  9. 根据权利要求7所述的应用，其特征在于，所述肿瘤为星形细胞瘤、间变性大细胞淋巴瘤、急性淋巴细胞白血病、急性骨髓性白血病、血管肉瘤、乳腺癌、B细胞淋巴瘤、膀胱癌、子宫颈癌、头颈癌、慢性淋巴细胞性白血病、慢性骨髓性白血病、结肠癌、直肠癌、子宫内膜癌、神经胶质瘤、胶质母细胞瘤、胃癌、胃泌素瘤、肝母细胞瘤、肝细胞癌、霍奇金淋巴瘤、卡波西肉瘤白血病、肺癌、平滑肌肉瘤、喉鳞状细胞癌、黑色素瘤、粘膜相关淋巴组织B细胞淋巴瘤、髓母细胞瘤、套细胞淋巴瘤、脑膜瘤、骨髓性白血病、多发性骨髓瘤、高危骨髓增生异常综合征、间皮瘤、神经纤维瘤、非霍奇金淋巴瘤、非小细胞肺癌、卵巢癌、食管癌、口咽癌骨肉瘤、胰腺癌、乳头状癌、前列腺癌、嗜铬细胞瘤、横纹肌肉瘤、头颈鳞状细胞癌、神经鞘瘤、小细胞肺癌、唾液腺肿瘤、散发性乳头状癌、 甲状腺癌、睾丸肿瘤或尿路上皮癌。
  10. 一种抗肿瘤的药物，其特征在于，包括活性链和互补链；
    所述活性链为权利要求1或5所述的RNA分子；所述互补链为权利要求2～5任一项所述的RNA分子。
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