CN106636368B - miR-130a在卵巢癌的诊断、治疗及预后中的应用 - Google Patents
miR-130a在卵巢癌的诊断、治疗及预后中的应用 Download PDFInfo
- Publication number
- CN106636368B CN106636368B CN201611065025.5A CN201611065025A CN106636368B CN 106636368 B CN106636368 B CN 106636368B CN 201611065025 A CN201611065025 A CN 201611065025A CN 106636368 B CN106636368 B CN 106636368B
- Authority
- CN
- China
- Prior art keywords
- mir
- ovarian cancer
- tsc1
- cells
- signaling pathway
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 206010033128 Ovarian cancer Diseases 0.000 title abstract description 52
- 206010061535 Ovarian neoplasm Diseases 0.000 title abstract description 52
- 238000011282 treatment Methods 0.000 title abstract description 13
- 238000003745 diagnosis Methods 0.000 title abstract description 7
- 238000004393 prognosis Methods 0.000 title abstract description 7
- 239000003112 inhibitor Substances 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000005557 antagonist Substances 0.000 claims description 4
- 239000000556 agonist Substances 0.000 claims 1
- 230000019491 signal transduction Effects 0.000 abstract description 33
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 abstract description 23
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 abstract description 23
- 230000004900 autophagic degradation Effects 0.000 abstract description 15
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 230000009545 invasion Effects 0.000 abstract description 7
- 230000004791 biological behavior Effects 0.000 abstract description 4
- 230000003211 malignant effect Effects 0.000 abstract description 4
- 230000035755 proliferation Effects 0.000 abstract description 4
- 201000011510 cancer Diseases 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 108020004999 messenger RNA Proteins 0.000 abstract description 3
- 238000013519 translation Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 230000009400 cancer invasion Effects 0.000 abstract 1
- 230000004614 tumor growth Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 59
- 102100031561 Hamartin Human genes 0.000 description 39
- 101000795643 Homo sapiens Hamartin Proteins 0.000 description 39
- 230000014509 gene expression Effects 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 19
- 108010057466 NF-kappa B Proteins 0.000 description 14
- 102000003945 NF-kappa B Human genes 0.000 description 14
- 239000002679 microRNA Substances 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 230000037361 pathway Effects 0.000 description 10
- 108700011259 MicroRNAs Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000002018 overexpression Effects 0.000 description 9
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 9
- 229960002930 sirolimus Drugs 0.000 description 9
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 9
- 238000001262 western blot Methods 0.000 description 8
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 7
- 238000011161 development Methods 0.000 description 7
- 230000018109 developmental process Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 5
- VSWDORGPIHIGNW-UHFFFAOYSA-N Pyrrolidine dithiocarbamic acid Chemical compound SC(=S)N1CCCC1 VSWDORGPIHIGNW-UHFFFAOYSA-N 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 102100040247 Tumor necrosis factor Human genes 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 108091070501 miRNA Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 108020005345 3' Untranslated Regions Proteins 0.000 description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 206010027476 Metastases Diseases 0.000 description 4
- 102000038030 PI3Ks Human genes 0.000 description 4
- 108091007960 PI3Ks Proteins 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 210000004957 autophagosome Anatomy 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 230000009401 metastasis Effects 0.000 description 4
- 210000003101 oviduct Anatomy 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 102000003954 Autophagy-Related Proteins Human genes 0.000 description 3
- 108010082399 Autophagy-Related Proteins Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000002611 ovarian Effects 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 102000000905 Cadherin Human genes 0.000 description 2
- 108050007957 Cadherin Proteins 0.000 description 2
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 2
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 101710175981 Hamartin Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 102100035100 Transcription factor p65 Human genes 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 230000006020 chronic inflammation Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102100033714 40S ribosomal protein S6 Human genes 0.000 description 1
- 101710107638 40S ribosomal protein S6 Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 230000007730 Akt signaling Effects 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- XYZZKVRWGOWVGO-UHFFFAOYSA-N Glycerol-phosphate Chemical compound OP(O)(O)=O.OCC(O)CO XYZZKVRWGOWVGO-UHFFFAOYSA-N 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027458 Metastases to lung Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150097381 Mtor gene Proteins 0.000 description 1
- 108050000637 N-cadherin Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241000233805 Phoenix Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 206010056342 Pulmonary mass Diseases 0.000 description 1
- 108010034782 Ribosomal Protein S6 Kinases Proteins 0.000 description 1
- 102000009738 Ribosomal Protein S6 Kinases Human genes 0.000 description 1
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108060008747 Ubiquitin-Conjugating Enzyme Proteins 0.000 description 1
- 102000003431 Ubiquitin-Conjugating Enzyme Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000035572 chemosensitivity Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000011500 cytoreductive surgery Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000522 effect on autophagocytosis Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000011990 functional testing Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108091040751 miR-130a stem-loop Proteins 0.000 description 1
- 108091041344 miR-130a-1 stem-loop Proteins 0.000 description 1
- 108091091365 miR-130a-2 stem-loop Proteins 0.000 description 1
- 108091031479 miR-204 stem-loop Proteins 0.000 description 1
- 108091079021 miR-27a stem-loop Proteins 0.000 description 1
- 108091043371 miR-27a-1 stem-loop Proteins 0.000 description 1
- 108091046123 miR-27a-2 stem-loop Proteins 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Hospice & Palliative Care (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种miR‑130a在卵巢癌的诊断、治疗及预后中的应用，本发明通过体内和体外功能试验明确miR‑130a通过抑制TSC1mRNA的翻译，使得mTOR信号通路过表达，从而促进卵巢癌细胞的增殖和侵袭，并且抑制了细胞的自噬。以上数据证明，降低内源性的miR‑130a可以抑制卵巢癌肿瘤的生长、侵袭等恶性生物学行为。基于此，可以设计特异性的miR‑130a抑制剂，恢复体内TSC1的含量，抑制mTOR信号通路，从而达到治疗卵巢癌的目的。
Description
技术领域
本发明涉及分子生物学领域，具体涉及一种miR-130a在卵巢癌的诊断、治疗及预后中的应用。
背景技术
卵巢癌是妇科常见恶性肿瘤，病死率一直位居妇科恶性肿瘤之首。虽然肿瘤细胞减灭术和铂类及紫杉类联合化疗使患者5年生存率有所改善，但半个世纪以来卵巢癌临床诊治并无突破性进展。究其根本原因在于对卵巢癌的发生发展的分子机制认识不清，缺乏临床实用的早期诊断手段和有效的治疗策略。因此，阐明卵巢癌发生发展的分子机制以及筛选与肿瘤发生发展和转移相关的分子标记物，对于其早期诊断和新防治靶点的确立具有重要价值。
大量研究表明PI3K/Akt-mTOR信号通路影响基因表达调控、细胞周期调控、细胞增殖及分化等。PI3K/Akt信号通路与多种癌症相关,包括黑色素瘤、乳腺癌、肺癌、胃癌、前列腺癌和胰腺癌。位于通路核心位置的TSC1/TSC2复合物起着极其重要的作用，该复合物是由TSC1的基因编码hamartin蛋白，马铃薯球蛋白(TSC2)交互形成，抑制信号转导的下游效应的哺乳动物雷帕霉素靶(MTOR)，从而抑制细胞的分裂增殖。大量文献资料显示TSC1基因在多种癌症中出现下调的趋势，如子宫内膜癌，乳腺癌。
microRNA(miRNA)是一类参与基因转录后调控的非编码小分子RNA，参与人体内包括肿瘤发生在内的多种生物学过程。近年来，miRNA的各种功能角色和相应机制在不断报道中。
发明内容
本发明基于对TSC1/TSC2复合物的研究认识，TSC2的3＇UTR仅仅为102bp，而TSC2的3＇UTR为4.8k，所以TSC1更容易受到microRNA的调控。过生物信息学分析，初步确定候选miRNA，瞬时转染miRNA mimics和inhibitors，通过western blot和Luciferase等方法明确靶向调控TSC1的miRNA；western blot检测miRNA对mTOR信号通路关键分子表达的影响；建立miRNA过表达和低表达的卵巢癌细胞系，研究miRNA及TSC1对卵巢癌细胞增殖、转化、侵袭能力及化疗敏感性的影响；最后通过动物实验明确靶向调控TSC1的miRNA与卵巢癌发生发展的关系。
通过以上研究方法，本发明发现：miR-130a在卵巢癌患者中高表达，同时与TSC1具有明显的负相关性，具有卵巢癌分子标记物的潜力。
因此，本发明提供了以下具体技术方案：
本发明的第一个方面，提供一种检测miRNA-130a含量的试剂盒在制备对受试者的卵巢癌进行诊断、预后评估或治疗效果监测的药物中的应用。
其中，所述检测对象为受试者的卵巢组织，所述受试者为人。
一种用于诊断或预后评估卵巢癌的试剂盒，该试剂盒中包括能定量检测miR-130a的试剂。
其包括miR-130a前引物：5’-CAGTGCAATGTTAAAAGGGCAT-3’如SEQ ID NO：20所示，以及通用后引物。所述通用后引物，本领域技术人员可以常规合成。
关于卵巢癌的诊断、预后评估、治疗效果监测，其方法包括：检测受试者卵巢组织中miR-130a的表达量、TSC1的含量，以及miR-130a的表达量和TSC1的含量在时间上的变化(如不同卵巢癌发展时期，治疗前和治疗后等)。根据检测结果，可以将受试者分为卵巢癌或健康，治疗反应好和坏，预测存活期长和短等。例如：可以将受试者的miR-130a的表达量、TSC1的含量与来自健康受试者的参比值比较，如果miR-130a表达量明显高于miR-130a表达量参比值，而TSC1的含量明显低于TSC1含量参比值，则该患者被高度怀疑为卵巢癌。
本领域中，TSC1基因编码hamartin蛋白，与马铃薯球蛋白(TSC2)交互形成复合物，该复合物为mTOR信号通路的重要的抑制剂。
本发明的第二个方面，提供一种miR-130a抑制剂在制备治疗卵巢癌的药物中的应用。
其中，所述miR-130a抑制剂是指抑制卵巢组织内miR-130a的表达水平的试剂。
进一步的，目前较为成熟的miR-130a抑制剂是指一段靶向于miR-130a的核苷酸序列，可以进入人体细胞内，靶向影响miR-130a的表达水平，目前合成工艺已相对完善。例如，该序列可为：5’-AUGCCCUUUUAACAUUGCACUG-3’，如SEQ ID NO：19所示，但不仅仅限于以上序列。
进一步的，miR-130a抑制剂在制备治疗通过抑制PI3K/Akt-mTOR信号通路的卵巢癌药物中的应用，其中，所述miR-130a的靶点为TSC1。
进一步的，可以利用NF-κB信号通路的拮抗剂，例如PDTC(吡咯烷二硫代氨基甲酸盐)，抑制该通路的激活，从而降低miR-130a在体内的含量。
本领域中技术人员知晓所述PI3K/Akt-mTOR信号通路，是指以mTOR复合物为主要成员的一个信号通路，活化的mTOR复合物可以磷酸化下游多个分子，例如40S核糖体蛋白S6激酶(S6K1)和真核细胞翻译起始因子4E-结合蛋白1(4E-BP1)从而促进蛋白质的合成和细胞的生长增殖。
本领域中技术人员知晓所述NF-kb信号通路，具体是指核因子-κB(Nuclearfactor-kappa B，NF-κB)是参与炎症过程的重要转录因子，目前是公认的炎症反应的关键性调节因子。在细胞内其与抑制蛋白IκB结合，以无活性的形式存在于细胞质中，当细胞受到炎性因子等刺激时，例如TNF-α即为该通路的一种特异性激活物，IκB发生磷酸化，从而被泛素结合酶识别而降解。NF-κB复合物被活化，进入细胞核，调节超过400种基因的转录，涉及到炎症，免疫调节，肿瘤细胞增殖、侵袭、转移、血管生成、耐药等多个方面。
本发明的第三个方面，提供一种miR-130a抑制剂，在制备卵巢癌细胞自噬诱导药物中的应用。
进一步的，miR-130a抑制剂联合雷帕霉素可以增强卵巢癌细胞自噬作用。
进一步的，miR-130a抑制剂在制备治疗通过抑制PI3K/Akt-mTOR信号通路的卵巢癌细胞自噬诱导药物中的应用，其中，所述miR-130a的靶点为TSC1。
进一步的，如上述所述，可以利用NF-κB信号通路的拮抗剂，例如PDTC(吡咯烷二硫代氨基甲酸盐)，抑制该通路的激活，从而降低miR-130a在体内的含量，从而达到卵巢癌的治疗的目的。
本发明通过western blot检测自噬相关的蛋白以及GFP-LC3单荧光体系示踪实验。分别比较miR-130a过表达和低表达对于细胞自噬的影响。雷帕霉素作为自噬的诱导剂而使用，本发明证明miR-130a抑制剂可以增强雷帕霉素的作用，使其自噬的效果增强。而miR-130a过表达后抑制了雷帕霉素所诱导的自噬现象。
本发明的第四个方面，提供一种miR-130a抑制剂在制备mTOR信号通路抑制剂中的应用。
经过研究发现，miR-130a抑制剂，能够恢复体内TSC1的含量，抑制mTOR信号通路，从而达到治疗卵巢癌的目的。
上述技术方案中的一个技术方案具有如下有益效果：
(1)为卵巢癌的诊断、病情评估和预后判断提供有效的分子标记物：本发明通过大量临床样本的验证，采用qPCR、western blot以及免疫组织化学染色等方法证明了miR-130a在卵巢癌患者中高表达，同时与TSC1具有明显的负相关性，具有卵巢癌分子标记物的潜力。
(2)为卵巢癌的诊断和治疗提供新的靶点：本发明通过体内和体外功能试验明确miR-130a通过抑制TSC1mRNA的翻译，使得mTOR信号通路过表达，从而促进卵巢癌细胞的增殖和侵袭，并且抑制了细胞的自噬。以上数据证明，降低内源性的miR-130a可以抑制卵巢癌癌灶的生长、侵袭等恶性生物学行为。基于此，可以设计特异性的miR-130a拮抗剂或抑制剂，恢复体内TSC1的含量，抑制mTOR信号通路，从而达到治疗卵巢癌的目的。
(3)本发明首次公布了miR-130a的靶点TSC1，TSC1作为mTOR信号通路的阀门具有重要的意义。本发明为mTOR信号通路的靶向调控找到了新的有效地方法，即通过miR-130amimics/inhibitor实现mTOR通路的激活和抑制。相比较于雷帕霉素抑制mTOR通路，此方法靶向性更强。
(4)本发明通过luciferase和CHIP等试验证明NF-kb可以结合到miR-130a的启动子区域，促进miR-130a的表达。从microRNA角度阐明了炎症在卵巢癌发生发展中的作用。即miR-130a为炎症通路NF-κB和mTOR信号通路之间的桥梁，我们可以认为慢性炎症通过激活NF-κB信号通路，促进miR-130a的高表达，miR-130a过表达后激活了mTOR通路，促进了卵巢癌的发生发展。
附图说明
图1是TSC1在卵巢癌组织中低表达的图。
图2是TSC1是miR-130a的直接靶基因的图。
图3是miR-130a提高细胞的增殖和侵袭能力的图。
图4是miR-130a抑制细胞的自噬的图。
图5是老鼠肺转移模型的构建的图。
图6是NF-kb信号通路促进miR-130a的表达的图。
图7是结论示意图。
具体实施方式
下面通过实施例对本发明进行具体的描述，有必要在此指出的是以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，该领域的技术人员可以根据上述本发明内容对本发明作出一些非本质的改进和调整。下述实施例中，若非特意表明，所用的试剂均为分析纯，所用试剂均可从商业渠道获得。文中未注明具体条件的实验方法，通常按照常规条件，或按照制造商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。
本发明中所使用引物如表1所示。
表1引物表
实施例1明确卵巢癌中miR-130a以及TSC1所扮演的角色
收集卵巢癌组织以及对照正常输卵管伞组织(组织均来源于山东大学齐鲁医院2009-2015)，并用组织研磨器冰上研磨，加入蛋白裂解液，冰上裂解20min，12000r/min4℃离心15min后取上清，BCA法测得蛋白浓度，并进行western blot和免疫组织化学染色的方法检测TSC1在卵巢癌组织和输卵管伞中的含量差异，结果显示在绝大多数卵巢癌组织中TSC1为低表达的(图1a)，灰度分析可以看出相比于正常输卵管伞组织，卵巢癌组织中TSC1的含量较低，两者有显著的统计学差异(p＜0.0001)(图1b)随后我们对含有43例输卵管伞组织和109例卵巢癌组织的组织芯片进行免疫组织化学染色，图1c为FT和HGSOC的典型染色图，对染色情况进行评分并统计分析，结果显示TSC1在卵巢癌中低表达(图1d)，且与卵巢癌的分期有关(图1e)即：FIGO分期越晚，TSC1含量越低，miR-130a则在卵巢癌患者中卵巢癌组织高表达(图2d)，同时与TSC1在同一患者组织中存在负相关性(图2e)。
实施例2确定调控miR-130a对TSC1的靶向调控作用
miRNA和3′-UTR的表达构建：
从吉玛基因公司购买miR-130a-3p、miR-27a-3p和miR-204-3p mimics以及阴性对照。miR-130a-inhibitor(是指序列5’-AUGCCCUUUUAACAUUGCACUG-3’，如SEQ ID NO：18所示)和阴性对照。瞬时转染卵巢癌细胞系，发现只有在转染了miR-130a后TSC1含量降低(图2a)同时我们发现miR-130a过表达后mTOR信号通路的关键蛋白的磷酸化水平明显升高，说明miR-130a激活了mTOR信号通路，反之miR-130a低表达后，mTOR信号通路被抑制(图2b)即miR-130a与mTOR信号通路的激活程度呈正相关。为了进一步的验证TSC1是miR-130a的直接靶基因，我们将TSC1 3’UTR区951bp序列通过Pmel和Xhol连接到pmirGLO(Promega)质粒上，构建野生型质粒。突变型质粒由生工生物工程(上海)股份有限公司合成。将野生型质粒和突变型质粒与miR-130a/NC共转染A2780细胞系，48小时后裂解细胞，进行荧光检测。结果如图2c miR-130a可以有效地抑制野生型质粒的荧光表达，但是不能抑制突变型的荧光表达，说明TSC1是miR-130a的靶基因。
实施例3构建miR-130a过表达的细胞系。
将miR-130a的序列连接到pGIPZ质粒(可常规购买得到)上，慢病毒包装后转染A2780和SKOV3细胞，利用嘌呤霉素进行筛选。筛选后用western blot和qPCR检测miR-130a和TSC1的含量。细胞系构建成功后用平板克隆实验和软琼脂克隆形成实验检测细胞的增殖能力，以及Transwell实验检测细胞的侵袭能力。
1、稳转细胞系构建：
(1)病毒颗粒的产生
取状态良好的Phoenix amphotropic细胞，胰酶消化收集细胞并计数后按照3×106个细胞/皿的密度接种于100mm细胞培养皿中，转染前的细胞培养液不能含有抗生素，放入细胞培养箱中培养过夜，观察当细胞密度达到70％-80％时进行质粒转染；
配制转染复合物：用1.6ml无血清的OPTI-MEM培养基分别稀释10μg的质粒DNA和36μl的脂质体,吹匀后室温孵育5min；
将两者混合，吹匀并避免产生气泡，室温孵育20min左右以形成DNA-脂质体混合物；
将上述DNA-脂质体复合物加入到铺好细胞的培养皿中置于培养箱中；
培养3h后取出，往培养皿中补加5ml OPTI-MEM培养基，继续培养6h左右之后换以6ml新鲜的无抗生素的DMEM完全培养基，放回培养箱中继续培养；
培养24h后收集含有病毒颗粒的上清液，暂存于4℃，并向皿中加入6ml新鲜完全培养基；
培养48h后再次收集上清并将两次收集的上清液混合，20000rpm，4℃离心2h，获得病毒颗粒沉淀；
用500μl培养基重悬病毒沉淀，进行下一步实验或储存于-80℃。
(2)感染宿主细胞
将待感染细胞按照3×104个细胞/ml的密度接种到6孔板中，培养过夜；
铺板第二天，每孔加入含8μg/ml Polybreen(及50μl浓缩的病毒颗粒进行感染。晃动培养板(十字晃)使其混匀。放入培养箱中继续培养；
感染48h后，吸弃上清，更换含有2μg/ml嘌呤霉素的培养基进行筛选，每隔2天观察细胞生长状态，每3-4天更换一次含有嘌呤霉素的培养液，持续筛选2周后即可得到稳定转染目的质粒的细胞。
2、平板克隆：选取对数生长期细胞，膜蛋白酶消化，细胞计数，400个细胞每孔，接种于6孔板中，培养约10-14天，甲醇固定15min，结晶紫染色30min。镜下观察并进行计数。结果见图3a，我们可以观察到miR-130a过表达后细胞的克隆形成能力明显增强。
3、Transwell:配制Matrigel和DMEM的混合液，其体积比为1:10，每孔加入50μl,该混合液待胶凝固后，上室加入200μl细胞与无血清DMEM混悬液(A2780 20万/孔，SKOV3 10万/孔)，下室加入含有10％FBS的DMEM，培养48小时后，甲醇固定，结晶紫染色后镜下观察计数。
结果见图3c，miR-130a过表达细胞系的侵袭能力明显强于对照组。同时我们检测了侵袭相关的EMT通路中的关键蛋白，结果如图3d，miR-130a过表达后细胞内N-cadherin和Vimentin含量增加，而E-cadherin含量降低，此结果进一步证明了miR-130a可以促进细胞的侵袭能力。
4、软琼脂克隆形成实验：下层胶：1％琼脂糖胶：2×DMEM(20％FBS)＝4:1，上述混合液3mL加入6cm皿中，4℃凝固后取出。上层胶：0.7％琼脂糖胶：2×DMEM(20％FBS)＝1:1，细胞5000个/皿，细胞稀释于50ul培养基中，上述混合液和细胞悬液共同加入培养皿中。4℃凝固，在5min中以内取出，并置于细胞培养箱中，10-14天进行结晶紫染色并观察计数。结果如图3b，miR-130a过表达后细胞在软琼脂中克隆形成的能力明显增强。
综上所述，miR-130a可以促进细胞的增殖能力，克隆形成能力以及侵袭能力。
实施例4细胞自噬
通过western blot检测自噬相关的蛋白以及GFP-LC3单荧光体系示踪实验分别比较miR-130a过表达和低表达对于细胞自噬的影响。雷帕霉素作为自噬的诱导剂而使用，通过western blot和灰度分析检测自噬相关的分子标记物，我们证明，miR-130a过表达后抑制细胞自噬(图4a-b)同时我们发现miR-130a inhibitor可以增强雷帕霉素的作用，使其自噬的效果增强(图4c-d)。
GFP-LC3单荧光体系示踪实验
取生长状态良好，处于对数生长期，汇合度约为70％-80％的A2780GFP-LC3细胞(为稳定转染GFP-LC3质粒的A2780细胞)，胰酶消化收集细胞，按照3×104个细胞/ml的密度将细胞接种于含有飞片(20mm)的六孔板中；培养过夜后，吸弃上清，实验组换以含10μM甲基莲心碱的新鲜培养液；对照组则换以含有等量于10μM甲基莲心碱剂量的DMSO的培养液，37℃，5％CO2恒温培养箱中继续培养24h；吸弃上清，用PBS洗涤细胞2次，用75％磷酸甘油固定细胞并防止干片，于荧光显微镜下观察GFP-LC3融合蛋白的聚集情况并拍照；自噬小体的量化采用计数每个细胞中含有GFP-LC3自噬小体的平均数，从任意10个视野中计数至少100个细胞中含有GFP-LC3自噬小体的平均数。结果见图4e，在90nM雷帕霉素处理下miR-130a过表达的细胞内自噬小体的数目明显少于对照组，而在60nM雷帕霉素处理下，miR-130a低表达的细胞内自噬小体明显增多。即miR-130a可以抑制细胞自噬。
实施例5 NOD-Prkdcem26Cd52/Nju雌鼠肺转移模型的构建
8-10周龄NOD-Prkdcem26Cd52/Nju雌鼠对照和实验组各5只。对照组注射SKOV3-pGIPZ-NC细胞，实验组注射SKOV3-pGIPZ-miR-130a细胞，每只注射500万细胞，细胞稀释于120μlPBS中，于尾部静脉进行注射。8周后麻醉处死老鼠，取肺部浸泡于10％福尔马林溶液中，后石蜡包埋，然后进行切片，HE染色和免疫组织化学染色以观察肺部结节的形态和数目。
1、HE染色步骤
烤片：60度烤片30min。
脱蜡：二甲苯I15min——二甲苯15min——100％酒精5min——100％酒精5min——95％酒精5min——80％酒精5min——75％酒精5min，自来水中涮洗2-3次。
苏木素染色10min，用小水流轻轻冲洗玻片，注意：水流过大可将组织冲走。
盐酸酒精1-2秒，氨水5-10秒，自来水中涮洗2-3次。
伊红染色20min，自来水中涮洗2-3次。
脱水：75％酒精10s——80％酒精10s——95％酒精10s——100％酒精10s——100％酒精10s——二甲苯I3min——二甲苯3min。
2、免疫组织化学染色
脱蜡步骤同上。
抗原修复：加入修复液(柠檬酸钠)附有组织的一面朝上，置于洗涤盒中，微波高火加热40s，待出现沸腾，暂停30秒。此时进入15个循环的蒸煮。12秒open+48秒close，冷却至室温(30min-60min)；湿盒中3％过氧化氢37度孵育15-20min，PBS摇床洗3次*3min；血清封闭：滴加试剂A(蓝色液体)37度孵育25-30min；加一抗：室温2小时，加试剂B：37度孵育15-20min，PBS摇床洗3次*3min；加试剂B：37度孵育10-15min，PBS摇床洗3次*3min。
加显色液DAB，苏木素染色，盐酸酒精中浸泡2秒---氨水中7-10秒；进行脱水(步骤同上)。
图5a为裸鼠肺部HE和免疫组织化学染色图像，我们可以观测到注射miR-130a过表达细胞系的裸鼠，肺内转移灶更多，且肿瘤体积更大，肿瘤细胞内TSC1染色浅。与对照组相比有显著的统计学差异(图5b)。说明miR-130a促进卵巢癌细胞系的恶性生物学行为。
实施例6明确NF-kb与miR-130a的关系
我们发现炎性因子LPS可以促进miR-130a的表达水平，在加入PDTC(NF-κB信号通路的抑制剂)以后，LPS便很难引起miR-130a的高表达(图6a)，说明LPS可能是通过NF-κB信号通路发挥作用的，同时NF-κB信号通路特异性的激活剂TNF-α可以促进miR-130a的表达(图6b)，western blot也进一步证明TNF-α在激活NF-κB信号通路的同时激活了mTOR信号通路(图6c)，为了进一步验证NF-kb信号通路是miR-130a的上游通路，我们进行了Luciferase实验，pGL4.20-TATA-miR-130a-promoter-F1/F2是由浙江大学赵斌教授赠送，也可按照本领域的技术手段常规合成，F1/F2在miR-130a启动子区域所处的位置见图6d。结果见图6e，我们可以检测到含有F2片段的质粒在加入TNF-α后luciferase荧光可以被显著增强，说明miR-130a的启动子区域存在NF-kB的结合位点，主要分布在F2区域。同时，我们进行了CHIP实验确定miR-130a启动子区域存在NF-kb的结合位点。
CHIP：
A780细胞种于10cm皿中，TNF-α(1ng/ml)处理2h后用进行交联，裂解，超声将染色体断裂为200-500bp大小的片段。
根据组别加入免疫沉淀抗体：
阳性对照，RNA聚合酶抗体，每管加入1.0μg抗体；
阴性对照，鼠IgG抗体，每管加入1.0μg抗体；
实验组：NF-kb p65抗体，每管加入5μg；
Rotator，4℃过夜。
每管加入60μL的Protein G Agarose，Rotator上，4℃旋转1-2h。清洗珠子，并进行protein/DNA复合体的洗脱，解交联释放DNA。然后进行qPCR检测预测位点的含量，从而确定NF-kb与miR-130a启动子区域结合的位点。结果表明，p65的结合位点包括位点1、3和4，目前我们可以确定miR-130a的启动子区域有NF-κB的结合位点，包括位点1、3和4，但是我们不能排除还有其他结合位点的可能性，因为预测软件存在局限性。
综上所述，本发明的原理可以阐述为：慢性炎症所导致的炎性因子的异常表达(如TNF-α)可以激活NF-κB信号通路，促进NF-κB复合物进入细胞核，结合到miR-130a的启动子区域，促进miR-130a的高表达，而miR-130a通过抑制TSC1的表达激活了mTOR信号通路，促进细胞的增殖和侵袭，即细胞的恶性生物学行为增强，最终发展为卵巢癌。(见图7)
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 山东大学
<120> miR-130a 在卵巢癌的诊断、治疗及预后中的应用
<130> 2016
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> 人工序列
<400> 1
cagtgcaatg ttaaaagggc at 22
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<400> 2
ttccctttgt catcctatgc ct 22
<210> 3
<211> 21
<212> DNA
<213> 人工序列
<400> 3
ttcacagtgg ctaagttccg c 21
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<400> 4
gcttcggcag cacatatact aaaat 25
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<400> 5
caacaagcaa atgtcgggga g 21
<210> 6
<211> 19
<212> DNA
<213> 人工序列
<400> 6
catagggcca cggtcagaa 19
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<400> 7
tgcaccacca actgcttagc 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列
<400> 8
ggcatggact gtggtcatga g 21
<210> 9
<211> 24
<212> DNA
<213> 人工序列
<400> 9
atctccagcc aatcccacca tttc 24
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<400> 10
aggaagcagc atctgagact tgaa 24
<210> 11
<211> 22
<212> DNA
<213> 人工序列
<400> 11
tcaacctgct gttctgactg tc 22
<210> 12
<211> 22
<212> DNA
<213> 人工序列
<400> 12
gagaagggag ctgagataca cc 22
<210> 13
<211> 24
<212> DNA
<213> 人工序列
<400> 13
ggatgtgatg gcacgtacct gaag 24
<210> 14
<211> 25
<212> DNA
<213> 人工序列
<400> 14
tggagacaag gtcttgatgt gttgc 25
<210> 15
<211> 22
<212> DNA
<213> 人工序列
<400> 15
ggtgaggctg aaaggctgag tg 22
<210> 16
<211> 23
<212> DNA
<213> 人工序列
<400> 16
gcaggtgaag gcagatccag ttc 23
<210> 17
<211> 31
<212> DNA
<213> 人工序列
<400> 17
catgtttaaa caaggcattg attgcacttg a 31
<210> 18
<211> 29
<212> DNA
<213> 人工序列
<400> 18
gccctcgagt gtagtgcaaa acaataaat 29
<210> 19
<211> 22
<212> RNA
<213> 人工序列
<400> 19
augcccuuuu aacauugcac ug 22
<210> 20
<211> 22
<212> DNA
<213> 人工序列
<400>20
cagtgcaatg ttaaaagggc at 22
Claims (1)
1.miR-130a拮抗剂或抑制剂在制备TSC1激动剂中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611065025.5A CN106636368B (zh) | 2016-11-28 | 2016-11-28 | miR-130a在卵巢癌的诊断、治疗及预后中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611065025.5A CN106636368B (zh) | 2016-11-28 | 2016-11-28 | miR-130a在卵巢癌的诊断、治疗及预后中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106636368A CN106636368A (zh) | 2017-05-10 |
| CN106636368B true CN106636368B (zh) | 2020-07-21 |
Family
ID=58811664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611065025.5A Active CN106636368B (zh) | 2016-11-28 | 2016-11-28 | miR-130a在卵巢癌的诊断、治疗及预后中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636368B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108342355B (zh) * | 2018-01-10 | 2021-06-18 | 南京艾维艾康生物技术有限公司 | 原始卵泡激活剂及其在人卵巢皮质培养液中的应用 |
| WO2020171889A1 (en) * | 2019-02-19 | 2020-08-27 | University Of Rochester | Blocking lipid accumulation or inflammation in thyroid eye disease |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105903036A (zh) * | 2015-07-15 | 2016-08-31 | 浙江大学 | miR-130a反义核酸及其衍生物在Hippo-YAP信号通路抑制剂中的应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101852805B (zh) * | 2009-03-31 | 2015-04-01 | 浙江大学 | Angptl3作为卵巢癌的诊断标记物的用途 |
-
2016
- 2016-11-28 CN CN201611065025.5A patent/CN106636368B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105903036A (zh) * | 2015-07-15 | 2016-08-31 | 浙江大学 | miR-130a反义核酸及其衍生物在Hippo-YAP信号通路抑制剂中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| Altered microRNA expression in cisplatin-resistant ovarian cancer cells and upregulation of miR-130a associated with MDR1/P-glycoprotein-mediated drug resistance;LINGYUN YANG 等;《ONCOLOGY REPORTS》;20121231;第28卷;第592页摘要、第593页右栏第3段、第599页右栏第2-3段 * |
| miR-130a在卵巢癌顺铂耐药细胞株中的表达及其意义;杨凌云 等;《四川大学学报（医学版）》;20121231;第43卷(第1期);第60页摘要、第61页第1.2.3节和表1、第64页左栏第3段和右栏第1段 * |
| NF-κB抑制剂PDTC对卵巢癌细胞HO8910增殖、凋亡的影响;孔祥 等;《扬州大学学报（农业与生命科学版）》;20080331;第29卷(第1期);第20页摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106636368A (zh) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN | |
| Sun et al. | Hsa-miR-139-5p inhibits proliferation and causes apoptosis associated with down-regulation of c-Met | |
| Zhang et al. | MicroRNA‐657 promotes tumorigenesis in hepatocellular carcinoma by targeting transducin‐like enhancer protein 1 through nuclear factor kappa B pathways | |
| Kan et al. | Chemokine (C‐C motif) ligand 5 is involved in tumor‐associated dendritic cell‐mediated colon cancer progression through non‐coding RNA MALAT‐1 | |
| Liu et al. | CCL5 promotes VEGF-dependent angiogenesis by down-regulating miR-200b through PI3K/Akt signaling pathway in human chondrosarcoma cells | |
| Qi et al. | OVOL2 links stemness and metastasis via fine-tuning epithelial-mesenchymal transition in nasopharyngeal carcinoma | |
| Wan et al. | miR-203 suppresses the proliferation and metastasis of hepatocellular carcinoma by targeting oncogene ADAM9 and oncogenic long non-coding RNA HULC | |
| Zhang et al. | Lnc-PDZD7 contributes to stemness properties and chemosensitivity in hepatocellular carcinoma through EZH2-mediated ATOH8 transcriptional repression | |
| Cheng et al. | miR-144-3p serves as a tumor suppressor by targeting FZD7 and predicts the prognosis of human glioblastoma. | |
| Wu et al. | Tumor cell–derived TGFβ1 attenuates antitumor immune activity of T cells via regulation of PD-1 mRNA | |
| Dong et al. | LncRNA COL1A1-014 is involved in the progression of gastric cancer via regulating CXCL12-CXCR4 axis | |
| Yu et al. | MicroRNA-10b induces vascular muscle cell proliferation through Akt pathway by targeting TIP30 | |
| Chen et al. | LncRNA growth arrest‐specific transcript 5 targets miR‐21 gene and regulates bladder cancer cell proliferation and apoptosis through PTEN | |
| Yang et al. | RETRACTED ARTICLE: Meg3 Induces EMT and Invasion of Glioma Cells via Autophagy | |
| Cao et al. | hsa_circ_0003410 promotes hepatocellular carcinoma progression by increasing the ratio of M2/M1 macrophages through the miR‐139‐3p/CCL5 axis | |
| Duan et al. | The long noncoding RNA ZFAS1 potentiates the development of hepatocellular carcinoma via the microRNA-624/MDK/ERK/JNK/P38 signaling pathway | |
| Yang et al. | DNMT1/miR-200a/GOLM1 signaling pathway regulates lung adenocarcinoma cells proliferation | |
| Xu et al. | Overexpression of long noncoding RNA H19 downregulates miR‐140‐5p and activates PI3K/AKT signaling pathway to promote invasion, migration and epithelial‐mesenchymal transition of ovarian cancer cells | |
| Zhang et al. | Overexpression of miR‐301a‐3p promotes colorectal cancer cell proliferation and metastasis by targeting deleted in liver cancer‐1 and runt‐related transcription factor 3 | |
| Liu et al. | MicroRNA-101-3p suppresses proliferation and migration in hepatocellular carcinoma by targeting the HGF/c-Met pathway | |
| Huang et al. | METTL1 promotes neuroblastoma development through m7G tRNA modification and selective oncogenic gene translation | |
| Wang et al. | Dysregulation of miR-6868-5p/FOXM1 circuit contributes to colorectal cancer angiogenesis | |
| Huang et al. | miR-150 contributes to the radioresistance in nasopharyngeal carcinoma cells by targeting glycogen synthase kinase-3β | |
| Niu et al. | Circ_0091579 promotes proliferative ability and metastasis of liver cancer cells by regulating microRNA-490-3p. | |
| Dong et al. | CircPCBP2 promotes the stemness and chemoresistance of DLBCL via targeting miR‐33a/b to disinhibit PD‐L1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |