CN114959033B - 一种外泌体piRNA的用途 - Google Patents
一种外泌体piRNA的用途 Download PDFInfo
- Publication number
- CN114959033B CN114959033B CN202210628527.3A CN202210628527A CN114959033B CN 114959033 B CN114959033 B CN 114959033B CN 202210628527 A CN202210628527 A CN 202210628527A CN 114959033 B CN114959033 B CN 114959033B
- Authority
- CN
- China
- Prior art keywords
- hsa
- pir
- small cell
- lung cancer
- cell lung
- 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
- 210000001808 exosome Anatomy 0.000 title claims abstract description 66
- 108091007412 Piwi-interacting RNA Proteins 0.000 title abstract description 13
- 239000004055 small Interfering RNA Substances 0.000 title abstract description 11
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims abstract description 88
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims abstract description 87
- 210000002966 serum Anatomy 0.000 claims abstract description 46
- 210000001519 tissue Anatomy 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 238000011156 evaluation Methods 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 6
- 238000004393 prognosis Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 238000001356 surgical procedure Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 80
- 230000014509 gene expression Effects 0.000 abstract description 39
- 239000000556 agonist Substances 0.000 abstract description 27
- 239000003112 inhibitor Substances 0.000 abstract description 25
- 238000000034 method Methods 0.000 abstract description 21
- 206010028980 Neoplasm Diseases 0.000 abstract description 20
- 201000011510 cancer Diseases 0.000 abstract description 17
- 238000004458 analytical method Methods 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 12
- 239000003550 marker Substances 0.000 abstract description 7
- 230000006870 function Effects 0.000 abstract description 4
- 108091027963 non-coding RNA Proteins 0.000 abstract description 4
- 102000042567 non-coding RNA Human genes 0.000 abstract description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 3
- 206010041067 Small cell lung cancer Diseases 0.000 abstract description 3
- 208000000587 small cell lung carcinoma Diseases 0.000 abstract description 3
- 230000003915 cell function Effects 0.000 abstract description 2
- 238000007619 statistical method Methods 0.000 abstract description 2
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 38
- 230000006907 apoptotic process Effects 0.000 description 17
- 230000005012 migration Effects 0.000 description 14
- 238000013508 migration Methods 0.000 description 14
- 230000035755 proliferation Effects 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 238000004445 quantitative analysis Methods 0.000 description 13
- 230000012292 cell migration Effects 0.000 description 12
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 11
- 229910052802 copper Inorganic materials 0.000 description 11
- 239000010949 copper Substances 0.000 description 11
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 239000000725 suspension Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000000605 extraction Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 239000000090 biomarker Substances 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 6
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 5
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 5
- 108010036226 antigen CYFRA21.1 Proteins 0.000 description 5
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 5
- 201000005202 lung cancer Diseases 0.000 description 5
- 208000020816 lung neoplasm Diseases 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 229940104230 thymidine Drugs 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- CDEURGJCGCHYFH-DJLDLDEBSA-N 5-ethynyl-2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(C#C)=C1 CDEURGJCGCHYFH-DJLDLDEBSA-N 0.000 description 4
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 4
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 4
- 238000002737 cell proliferation kit Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 108091032955 Bacterial small RNA Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- -1 rRNA Proteins 0.000 description 3
- 208000000649 small cell carcinoma Diseases 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 108050008874 Annexin Proteins 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010040476 FITC-annexin A5 Proteins 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 101001126085 Homo sapiens Piwi-like protein 1 Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- 102100029364 Piwi-like protein 1 Human genes 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100037904 CD9 antigen Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 101100408379 Drosophila melanogaster piwi gene Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 1
- 101000613251 Homo sapiens Tumor susceptibility gene 101 protein Proteins 0.000 description 1
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 description 1
- 108010066302 Keratin-19 Proteins 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108091092355 RNA of unknown function Proteins 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 102000039471 Small Nuclear RNA Human genes 0.000 description 1
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 1
- 102000042773 Small Nucleolar RNA Human genes 0.000 description 1
- 241001122767 Theaceae Species 0.000 description 1
- 102100040879 Tumor susceptibility gene 101 protein Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- MHMCOSLCKPWGQG-UHFFFAOYSA-H [U+6].[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O Chemical compound [U+6].[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O.[O-]C(=O)C([O-])=O MHMCOSLCKPWGQG-UHFFFAOYSA-H 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000002487 multivesicular body Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000003325 tomography Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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
-
- 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
- C12N15/1135—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 against oncogenes or tumor suppressor genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q2600/158—Expression 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
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明属于生物医学技术领域,具体涉及一种外泌体piRNA的用途,将血清来源的外泌体非编码RNA piR‑hsa‑164586用于非小细胞肺癌的早期检测和寻找用于克服癌症的潜在靶点,通过探究RNApiR‑hsa‑164586对非小细胞肺癌细胞系功能的影响,提高非小细胞肺癌的临床检测性能,具体过程是:首先,从组织、细胞和血清外泌体三个方面筛选出在非小细胞肺癌组织中显著高表达的RNApiR‑hsa‑164586;然后,通过构建ROC曲线对RNA piR‑hsa‑164586的检测性能进行统计分析;最后,将RNA piR‑hsa‑164586的表达与非小细胞肺癌临床特征进行关联性比较和分析,进一步探究RNApiR‑hsa‑164586的激动剂和抑制剂对非小细胞功能的影响,为非小细胞肺癌的早期检测提供了一种新型的血清标志物,为克服小细胞肺癌提供了潜在的治疗靶点。
Description
技术领域:
本发明属于生物医学技术领域,具体涉及一种外泌体piRNA的用途,作为早期检测非小细胞肺癌的试剂盒,以提高非小细胞肺癌的检测性能,寻找克服癌症的潜在靶点。
背景技术:
肺癌是癌症致死的首要原因。根据美国癌症协会2021年的癌症数据分析显示,提高癌症诊断和治疗手段是肺癌死亡率下降的主要原因。肺癌包括小细胞肺癌和非小细胞肺癌,其中,非小细胞肺癌的占比为80%-85%,由于癌症早期临床症状不明显且易与其他肺部疾病混淆,所以大约75%的患者被确认时已处于中晚期,5年生存率很低。目前,肺癌临床常用的检测方法包括组织病理活检技术、电子计算机 X射线断层扫描技术和蛋白水平的血清生物标志物。但是,其存在有创,易引起并发症和假阳性率高等问题。基于非小细胞肺癌早期检测方法不佳,一旦进展为中晚期,将会对患者本身及家庭造成重担,所以,亟需研发设计一种新型的检测生物标志物提高早期癌症的确认效率,通过研究其在癌症发展过程中的具体机制,为克服癌症寻找新的靶点,从而减轻病痛造成的严重后果。
外泌体是由活细胞分泌的直径为50-150nm的多囊泡体,可以将装载的多种物质如蛋白质、脂类和RNA转运到细胞外基质中去,从而在细胞外环境中介导细胞间通讯。由于外泌体天然存在于包括血液、尿液和乳汁的多种体液成分中,所以,可以通过检测体液中外泌体包裹的内容物来对疾病进行检测和分析。
非编码RNA指的是从基因组上转录来的不编码成蛋白质,而是在 RNA水平上直接行使其生物学功能的RNA,包括已知功能的RNA,如 tRNA、rRNA、snRNA、snoRNA和microRNA等,以及未知功能的RNA,如与PIWI蛋白相互作用的RNA(PIWI interacting RNA,piRNA)。piRNA是一类长度集中在29-30nt、表达具有组织特异性、以高度特异链的方式与基因组相对应的单链小RNA,通过与Piwi亚家族蛋白结合形成的复合物行使其功能,其表达失调在疾病的发生发展中起着重要作用,如沉默转录基因过程,调节翻译和维持mRNA的稳定性等。已有研究显示:piRNA存在于肿瘤来源的体液环境中,其异常表达对肿瘤检测具有一定的参考价值。基于外泌体可以调节癌症细胞之间的信息通讯,piRNA的表达具有组织特异性且其异常表达对癌症的进展发挥着重要作用,将血清来源的外泌体piRNA作为生物标志物,应用于癌症早期检测,研究其在癌症发生发展过程中的具体机制,提高临床血清蛋白检测标志物的性能,寻找克服癌症的新型潜在靶点,从而促进基因类标志物向临床的转化。
发明内容:
本发明的目的在于克服现有技术存在的缺点,寻求设计一种外泌体piRNA的用途,作为检测非小细胞肺癌的生物标志物,寻找克服癌症的潜在靶点,提高癌症检测的效能。
为了实现上述目的,本发明涉及的外泌体piRNA的用途为作为检测非小细胞肺癌的生物标志物和克服癌症的潜在靶点;外泌体piRNA 为血清外泌体来源的,与PIWI蛋白作用的非编码RNA piR-hsa- 164586;piR-hsa-164586的核苷酸序列片段为TGAGAACTGAATTCCATAGGCTGC,引物为Human-piR-hsa-164586,其上游引物序列Human-piR-hsa-164586-F为CGCGTGAGAACTGAATTCCATA,下游引物序列Human-piR-hsa-164586-R为AGTGCAGGGTCCGAGGTATT。
本发明涉及的RNA piR-hsa-164586通过外泌体提取试剂盒从血清中提取;
通过TEM(透射电子显微镜)、NTA(纳米颗粒跟踪分析)和 Western-Blot(蛋白质印迹法)三种技术对提取的RNA piR-hsa- 164586的结构进行验证;
通过总RNA(总核糖核酸)抽提试剂(TRIZOL试剂)裂解细胞或外泌体,提取包含piR-hsa-164586中的总RNA,将总RNA反转录成 cDNA(互补脱氧核糖核酸),通过qRT-PCR(实时荧光定量逆转录聚合酶链反应)分析RNA piR-hsa-164586的表达量;
根据组织高通量测序得出:RNA piR-hsa-164586在非小细胞肺癌组织中的表达是显著上调的,具有潜在的生物学功能和调节的信号通路。
本发明涉及的RNA piR-hsa-164586的稳定性验证过程为:
将同一血清样本分为两组:提取外泌体组与外泌体耗竭组,通过 qRT-PCR分别检测两组的RNA piR-hsa-164586的表达量,根据表达量判断外泌体悬液中是否富集了RNApiR-hsa-164586;
将同一血清来源的RNA piR-hsa-164586分为两组:RNase A处理组与RNase A未处理组,通过qRT-PCR检测两组的RNA piR-hsa- 164586的表达量,根据表达量判断外泌体是否对所包裹的RNA piR- hsa-164586起保护作用;
将血清来源的RNA piR-hsa-164586在室温下分别放置0h、 12h、24h和48h,通过qRT-PCR检测在室温下放置不同时间后的RNA piR-hsa-164586的表达量,根据表达量判断血清来源的外泌体在室温下长期放置后的稳定性。
本发明涉及的RNA piR-hsa-164586的对非小细胞肺癌早期的检测效能的评估方法为:
通过构建ROC曲线(接收者操作特征曲线)将RNA piR-hsa- 164586与生物标志物CYFRA21-1的检测性能进行分析和比较。
本发明涉及的RNA piR-hsa-164586的对非小细胞肺癌术后的预后效果的评估方法为:
通过qRT-PCR技术和构建ROC曲线评估同一组织术前和术后一周血清来源外泌体的RNA piR-hsa-164586判断手术对非小细胞肺癌组织的干预效果。
本发明涉及的RNA piR-hsa-164586的对非小细胞肺癌临床特征的反映情况的评估方法为:
通过qRT-PCR技术评估血清来源外泌体的RNA piR-hsa-164586 的表达情况,结合肺癌的临床特征的相关性,进行综合分析。
本发明涉及的RNA piR-hsa-164586的靶向作用效果的验证过程为:
将RNA piR-hsa-164586转染入非小细胞肺癌A549和HCC2279细胞系中,通过CCK8试剂盒(细胞计数试剂盒)和Edu(胸苷(T)类似物)染色检测非小细胞肺癌细胞增殖指标,根据非小细胞肺癌细胞增殖指标判断RNA piR-hsa-164586对细胞增殖活力的影响;
通过Trannswell(侵袭实验)小室和划痕实验检测非小细胞肺癌细胞迁移指标,根据非小细胞肺癌细胞迁移指标判断RNA piR- hsa-164586对细胞迁移能力的影响;
通过流式细胞术检测凋亡指标,根据凋亡指标判断RNA piR- hsa-164586对细胞凋亡的影响。
本发明与现有技术相比,将血清来源的外泌体非编码RNA piR- hsa-164586用于非小细胞肺癌的早期检测和寻找用于克服癌症的潜在靶点,通过探究RNA piR-hsa-164586对非小细胞肺癌细胞系功能的影响,提高非小细胞肺癌的临床检测性能,具体过程是:首先,从组织、细胞和血清外泌体三个方面筛选出在非小细胞肺癌组织中显著高表达的RNApiR-hsa-164586;然后,通过构建ROC曲线对RNApiR-hsa-164586的检测性能进行统计分析;最后,将RNA piR-hsa- 164586的表达与非小细胞肺癌临床特征进行关联性比较和分析,进一步探究RNA piR-hsa-164586的激动剂和抑制剂对非小细胞功能的影响;基于大样本量的数据分析和反复实验验证,可信度高,性能显著,为非小细胞肺癌的早期检测提供了一种新型的血清标志物,为克服小细胞肺癌提供了潜在的治疗靶点。
附图说明:
图1为本发明实施1例涉及的组织高通量测序结果示意图。其中,a为差异表达基因聚类分析图,b为相关系数矩阵图。
图2为本发明实施2例涉及的RNA piR-hsa-164586结构验证的结果示意图,其中,a为外泌体透射电子显微镜图,b为外泌体纳米颗粒跟踪分析图,c为外泌体表面标记蛋白质印迹实验图。
图3为本发明实施3例涉及的RNA piR-hsa-164586结构稳定性的评估结果示意图,其中,a为验证血清中外泌体有无时piR-hsa- 164586的表达结果,b为验证血清外泌体piR-hsa-164586在有无核糖核酸酶环境下的表达量有无差异性结果,c为验证血清外泌体piR-hsa-164586在环境中放置不同时长后表达量的变化结果。
图4为本发明实施4例涉及的RNA piR-hsa-164586的提取和基因表达水平分析的结果示意图。
图5为本发明实施5例涉及的RNA piR-hsa-164586对Ⅰ期及早期非小细胞肺癌检测效能评估的结果示意图,其中,a为通过构建ROC 曲线比较piR-hsa-164586和细胞角蛋白19-片段对I期非小细胞肺癌的检测性能结果,b为通过构建ROC曲线比较piR-hsa-164586和细胞角蛋白19-片段对I-Ⅲ期非小细胞肺癌的检测性能结果。
图6为本发明实施6例涉及的非小细胞肺癌组织术前与术后RNA piR-hsa-164586表达情况比较的结果示意图,其中,a为血清外泌体 piR-hsa-164586在非小细胞肺癌组织术前及术后表达量的差异性结果,b为通过构建ROC曲线评估血清外泌体piR-hsa-164586作为非小细胞肺癌组织术后预后的指标之一的性能结果。
图7为本发明实施7例涉及的RNA piR-hsa-164586是否能够作为非小细胞肺癌组织术后预后情况评估指标的结果示意图,其中a为比较非小细胞肺癌组织不同分期时血清外泌体piR-hsa-164586的表达量结果,b为比较有无***转移的非小细胞肺癌组织血清外泌体 piR-hsa-164586的表达量结果,c为比较不同年龄的非小细胞肺癌组织血清外泌体piR-hsa-164586的表达量结果,d为比较不同性别的非小细胞肺癌组织血清外泌体piR-hsa-164586的表达量结果,e为比较不同病理亚型的非小细胞肺癌组织血清外泌体piR-hsa-164586的表达量结果,f为比较吸烟与不吸烟的非小细胞肺癌组织血清外泌体piR-hsa-164586的表达量结果。
图8为本发明实施例11涉及的非小细胞肺癌细胞A549和HCC2279 CCK8增殖活力的影响结果示意图。其中,a为通过细胞计数实验验 (CCK8)验证激动剂对A549细胞增殖活力的影响,b为通过细胞计数实验(CCK8)验证抑制剂对A549细胞增殖活力的影响,c为通过细胞计数实验验(CCK8)验证激动剂对HCC2279细胞增殖活力的影响,d 为通过细胞计数实验验(CCK8)验证抑制剂对HCC2279细胞增殖活力的影响。
图9为本发明实施例12涉及的非小细胞肺癌细胞A549和HCC2279 增殖活力Edu染色的影响结果示意图,其中,a为通过胸苷(T)类似物(5-ethynyl-2’-deoxyuridine,Edu)细胞增殖检测试剂盒验证激动剂和抑制剂对A549细胞增殖活力的影响,b为通过胸苷(T)类似物(5-ethynyl-2’-deoxyuridine,Edu)细胞增殖检测试剂盒验证激动剂和抑制剂对HCC2279细胞增殖活力的影响。
图10为本发明实施例12涉及的非小细胞肺癌细胞A549和HCC2279 增殖活力Edu染色分析的统计定量分析结果示意图,其中,a为通过胸苷(T)类似物(5-ethynyl-2’-deoxyuridine,Edu)细胞增殖检测试剂盒验证激动剂和抑制剂对A549细胞增殖的定量分析,b为通过胸苷(T)类似物(5-ethynyl-2’-deoxyuridine,Edu)细胞增殖检测试剂盒验证激动剂和抑制剂对HCC2279细胞增殖的定量分析。
图11为本发明实施例13涉及的T非小细胞肺癌细胞A549和HCC2279 Transwell小室迁移能力的影响结果示意图,其中,a为通过细胞迁移实验(Transwell)验证激动剂和抑制剂对A549细胞迁移的影响,b为通过细胞迁移实验(Transwell)验证激动剂和抑制剂对HCC2279细胞迁移的影响。
图12为本发明实施例13涉及的非小细胞肺癌细胞A549和HCC2279 Transwell小室迁移能力的统计定量分析结果示意图,其中,a为通过细胞迁移实验(Transwell)验证激动剂和抑制剂对A549细胞迁移的影响定量定量分析,b为通过细胞迁移实验(Transwell)验证激动剂和抑制剂对HCC2279细胞迁移的影响的定量分析。
图13为本发明实施例14涉及的划痕实验对非小细胞肺癌细胞 A549和HCC2279迁移能力的影响结果示意图,其中,a为通过划痕实验验证激动剂和抑制剂对A549细胞迁移的影响,b为通过划痕实验验证激动剂和抑制剂对HCC2279细胞迁移的影响。
图14为本发明实施例14涉及的划痕实验对非小细胞肺癌细胞 A549和HCC2279迁移能力的统计定量分析结果示意图,其中,a为通过划痕实验验证激动剂和抑制剂对A549细胞迁移影响的定量分析,b为通过划痕实验验证激动剂和抑制剂对HCC2279细胞迁移影响的定量分析。
图15为本发明实施例15涉及的通过流式细胞仪对非小细胞癌细胞A549和HCC2279凋亡的影响结果示意图,其中,a为通过凋亡试剂盒验证激动剂和抑制剂对A549细胞凋亡的影响,b为通过凋亡试剂盒验证激动剂和抑制剂对HCC2279细胞凋亡的影响。
图16为本发明实施例15涉及的通过流式细胞仪对非小细胞癌细胞A549和HCC2279凋亡的统计定量分析结果示意图,其中,a为通过凋亡试剂盒验证激动剂和抑制剂对A549细胞凋亡影响的定量分析,b为通过凋亡试剂盒验证激动剂和抑制剂对HCC2279细胞凋亡影响的定量分析。
具体实施方式:
下面通过实施例并结合附图对本发明作进一步说明。
实施例1:
本实施例涉及利用组织高通量测序(北京百迈客生物科技有限公司)的方法筛选出在非小细胞肺癌组织与相对应的癌旁组织中差异性显著且高表达的RNA piR-hsa-164586,通过GO(功能富集)分析法和KEGG(通路富集)分析法对RNA piR-hsa-164586的功能和涉及的信号通路进行分析,结果如图1所示,RNA piR-hsa-164586在非小细胞肺癌组织中是明显表达上调的。
实施例2:
本实施例涉及RNA piR-hsa-164586的提取及验证的过程:
收集血清样本,用外泌体提取试剂盒(翌圣,41202ES30)提取 RNA piR-hsa-164586,具体的提取方法如下:
预处理:
(1)将收集的常温血清样本置于冰上待用;当血清样本为冻存的样品时,取出后置于25℃的水浴中解冻,待完全融化后再置于冰上待用;
(2)取1mL血清/血液样品转移至离心管中,在4℃,3000×g 的条件下离心10min,弃沉淀,将上清液转移至新的离心管中;
(3)将转移的上清液于4℃,10000×g的条件下离心20min,弃沉淀,将上清液转移至新的离心管中;
外泌体分离:
(1)向预处理后得到的最终上清液中,加入4倍体积的1×PBS (磷酸缓冲盐溶液)充分混匀;
(2)加入41202-A试剂,盖紧离心管盖,涡旋振荡1min,在 4℃条件下静置2h,得到混合液;
41202-A试剂用量根据下表选择:
Serum/Plasma(血清/血浆)+PBS | 外泌体提取试剂(41202-A)用量 |
200μL+800μL | 200μL |
1.0mL+4.0mL | 1.0mL |
(3)将装有混合液的离心管于4℃,10000×g条件下离心60min,弃上清液,收集富含外泌体的离心沉淀物;
(4)吸取1×PBS溶液均匀吹打离心沉淀物,待其充分悬浮于 PBS后,将重悬液转移至新的1.5mL离心管中;
1×PBS溶液与血清样本的体积比为1:2.5;
(5)将含有重悬液的1.5mL离心管于4℃,12000×g条件下离心2min,弃沉淀,保留上清液,该上清液即为富含外泌体颗粒的溶液,分装后保存,避免反复冻融;
当该上清液具有明显沉淀时,重复步骤(4),重新进行洗涤和沉淀;富含外泌体颗粒的溶液能够于4℃条件下保存3天,于-80℃条件下长期保存。
分别通过以下三种实验对血清外泌体的特征进行验证:
TEM(透射电子显微镜)分析
将富含外泌体颗粒的溶液固定在载样铜网上;
(1)重悬exosome(外泌体)到50-100μl,2%的PFA(可溶性聚四氟乙烯)中,得到exosome悬液;
(2)将5μl exosome悬液加到Formvar-carbon(碳支持膜) 载样铜网上;将铜网的碳支持膜的膜面朝下置于exosome悬液上,每份exosome悬液匹配2-3个铜网;
(3)将100μl PBS加到封口膜上,用镊子将Formvar膜面朝下的铜网置于PBS液滴上清洗;
(4)将铜网置于50μl,1%的戊二醛液滴上5min;
(5)将铜网置于100μl,ddH2O(二次蒸馏水)中冲洗2min,共冲洗8次;
外泌体负染色处理及电镜检测
(1)将铜网置于50μl,pH值为7的草酸双氧铀液滴上5min;
(2)将铜网置于50甲基纤维素液滴上10min,冰上操作;
(3)将铜网置于不锈钢环上,用滤纸吸去多余液体;
(4)空气中干燥5-10min;
(5)将铜网置于盒子中,在80kV条件下拍摄电镜照片;
NTA(纳米粒子追踪分析)
(1)用去离子水清洗样本池;
(2)用聚苯乙烯微球(110nm)校准仪器;
(3)用1×PBS(Biological Industries,Israel)(生物公司,以色列)清洗样本池;
(4)用1×PBS(BI,Israel)(生物公司,以色列)稀释样本,进样检测;
蛋白免疫印迹法(Western blotting)
通过放射免疫沉淀测定裂解缓冲液(RIPA,美仑)提取的外泌体总蛋白,通过BCA比色法(总蛋白定量测试试剂盒)比色法调节蛋白浓度使其达到一致;
通过SDS-PAGE(聚丙烯酰胺凝胶电泳)电泳对蛋白样品进行分离,转至PVDF膜,5%脱脂奶粉封闭1小时,洗膜,孵一抗(CD9, CD81,TSG101),于4℃的冰箱中孵育过夜,洗膜,室温孵育二抗1 小时,洗膜,采用ECL化学发光法显影得到目的条带。
结果如图2所示,血清外泌体呈典型的茶托盘样结构,粒径在 50-100nm范围内,血清外泌体特异性表面标记蛋白CD9,CD81, TSG101表达阳性,表明从血清中提取的即为外泌体颗粒。
实施例3:
本实施例涉及RNA piR-hsa-164586稳定性的评估:
将250ul血清做两种处理:将血清外泌体提取试剂盒提取的外泌体作为实验组,将血清外泌体耗竭完的上清液作为对照组;
将100ul外泌体悬液分为两组:RNase A(翌圣,10405ES03)处理组和RNase A未处理组,处理组加入的RNase A的浓度为 2ug/ml;
将1ml血清平均分成4份分别在室温下放置0、12、24和48h;
分别通过qRT-PCR实验验证每组的RNA piR-hsa-164586的表达水平;
结果如图3所示,富含外泌体的血清样本中RNA piR-hsa- 164586的表达显著升高;经过RNase A处理和在室温下放置不同时间后的RNA piR-hsa-164586的表达水平都未发生显著变化,并且RNA piR-hsa-164586在室温下放置较长时间且不受环境中RNA酶的影响。
实施例4:
本实施例涉及RNA piR-hsa-164586的提取和基因表达水平的分析:
在每100ul外泌体悬液中加入750ul Trizol和20ul冰醋酸裂解外泌体,室温条件下作用15分钟,在裂解后的液体中加入200u1氯仿,盖紧管盖,涡旋混匀,于室温条件下静置3钟后,于4℃, 12000g/min条件下离心15分钟;
将上层水相液体转移至新的1.5ml离心管中,加入500uL异丙醇,涡旋混匀,于室温条件下静置10分钟后,于4℃,12000g/min 条件下离心10分钟;
丢弃上清液,保留底部白色沉淀,并加入1mL,75%的乙醇(DEPC 水配制),用手轻轻将白色沉淀弹起,于4℃,7500g/min条件下离心5分钟;
丢弃上清液,于室温条件下干燥10-15分钟后,加入30-40uL DEPC水(无核酸酶的水)重新溶解,检测RNA浓度后,将RNA置于- 80℃环境中保存或直接使用;
使用反转录试剂盒(诺唯赞,货号MR101-00),将提取的 1000ng总RNA反转录成cDNA,使用实时荧光定量PCR技术检测基因的表达水平;
结果如图4所示,RNA piR-hsa-164586在全期及早期非小细胞肺癌组织的RNApiR-hsa-164586中的表达是显著上调的。
实施例5:
本实施例涉及RNA piR-hsa-164586对Ⅰ期及早期非小细胞肺癌检测效能的评估:
将实验数据构建成ROC曲线,比较RNA piR-hsa-164586和 CYFRA21-1曲线下面积(AUC)、敏感度和特异性,进行评估;
结果如图5所示,RNA piR-hsa-164586与CYFRA21-1相较,在Ⅰ期时,piR-hsa-164586的AUC比CYFRA21-1高11.7%,敏感度和特异性分别高5.7%和6.1%;在全期时,RNApiR-hsa-164586的AUC (曲线下面积)比CYFRA21-1(细胞角蛋白19片段)高13.1%,敏感度和特异性分别高5.4%和7.8%。
实施例6:
本实施例涉及非小细胞肺癌组织术前与术后RNA piR-hsa-164586表达情况的比较:分别在术前及术后一周收取同一组织的血清,通过提取外泌体和外泌体中的RNA用qRT-PCR进行定量分析;
结果如图6所示,非小细胞肺癌组织在术后一周RNA piR-hsa- 164586的表达显著下降,表明RNA piR-hsa-164586在非小细胞肺癌组织术后的表达含量明显下降。
实施例7:
本实施例涉及RNA piR-hsa-164586是否能够作为非小细胞肺癌组织术后预后情况的一项评估指标;
结果如图7所示,RNA piR-hsa-164586对早期非小细胞肺癌组织术后有67.9%的评估性能,敏感度为58.8%,表明RNA piR-hsa- 164586有潜力成为评估非小细胞肺癌组织术后预后情况的一项评估指标。
实施例8:
本实施例涉及RNA piR-hsa-164586的表达情况与非小细胞肺癌临床特征的相关性分析;
结果如图8所示,RNA piR-hsa-164586的表达情况与非小细胞肺癌的肿瘤分期、病理分级和吸烟有一定的相关性。
实施例9:
本实施例涉及RNA piR-hsa-164586构建激动剂和抑制剂的过程:以RNA piR-hsa-164586基因为模板,采用引物Human-piR-hsa- 164586进行PCR扩增,生成双链RNA piR-hsa-164586,针对RNApiR-hsa-164586反义链进行3’端胆固醇修饰、3’端四个硫代骨架修饰、5’端两个硫代骨架修饰和全链甲氧基修饰(上海吉玛制药技术有限公司),完成激动剂和抑制剂的构建。
实施例10:
本实施例涉及将激动剂和抑制剂转染至非小细胞肺癌细胞的过程:
当非小细胞肺癌细胞传代至细胞密度达到30-50%时,利用 lipo3000将激动剂转染至非小细胞肺癌细胞;
将5μl(100pmol)激动剂加入含有95μl DMEM培养基的EP管中吹打混匀后,再将5μllipo3000加入含有95μl DMEM培养基的 EP管中吹打混匀,将二者以1:1的体积比混合,形成转染混合物,于室温条件下静置20分钟后,置于培养板内,48小时后进行基因和蛋白水平分析。
实施例11:
本实施例涉及非小细胞肺癌细胞A549和HCC2279 CCK8增殖活力分析过程:
培养非小细胞肺癌细胞,对实验组进行RNA piR-hsa-164586的转染,分别于转染0、1、2、3、4天后,实验组和对照组非小细胞肺癌细胞分别吸弃原有的培养基,用PBS冲洗非小细胞肺癌细胞两次,加入90μl新鲜培养基和10μl CCK8试剂(陶术,C0005)处理2小时,在450nm吸光度处检测OD值,结果如图9所示,OD值大小代表细胞密度,反映细胞增殖活力,RNApiR-hsa-164586构建的激动剂能够明显促进非小细胞肺癌细胞A549和HCC2279的增殖;RNApiR- hsa-164586构建的抑制剂能够明显抑制非小细胞肺癌细胞A549和 HCC2279的增殖。
实施例12:
本实施例涉及非小细胞肺癌细胞A549和HCC2279增殖活力Edu 染色分析过程:
培养非小细胞肺癌细胞,对实验组进行RNA piR-hsa-164586的转染,转染两天后,进行Edu染色分析细胞增殖情况(美仑, MA0424/MA0424-L):
(1)Edu标记细胞:以10μM的Edu工作液在37℃避光条件下孵育细胞2小时;
(2)细胞固定及促渗:去除培养基,加入质量百分比浓度为4%的中性多聚甲醛室温固定15-30分钟后,去除固定液,加入质量百分比浓度为0.5%的Triton X-100(曲拉通X-100)in PBS,室温条件下孵育20分钟,促渗;
(3)EdU检测:加入Click-iT(蛋白质反应缓冲液试剂盒)反应混合物,简短摇晃培养板以确保反应混合物均匀覆盖细胞,室温避光条件下孵育30分钟;
(4)DNA复染:用5μg/mL Hoechst 33342(活细胞染色液)溶液于室温避光条件下孵育15-30分钟,染核;
(5)PBS洗涤细胞2次,通过荧光显微镜拍摄照片,分析正在增殖的非小细胞肺癌细胞比例;
结果如图10和图11所示,RNA piR-hsa-164586构建的激动剂能够显著促进非小细胞肺癌细胞的生长,RNA piR-hsa-164586构建的抑制剂能够显著抑制非小细胞肺癌细胞的生长。
实施例13:
本实施例涉及非小细胞肺癌细胞A549和HCC2279 Transwell小室迁移能力的分析过程:
培养非小细胞肺癌细胞,对实验组进行RNA piR-hsa-164586的转染,转染两天后,在24孔Millicell室中进行迁移测定:将200 μl无血清培养基中的细胞(2×104)添加到包被的滤膜中,将含有 20%胎牛血清的500μl培养基作为化学吸引剂添加到下腔室中,在 37℃的恒温箱中放置24小时后,将通过滤膜迁移的细胞用甲醇固定,用0.5%的结晶紫染色,在显微镜下对细胞数进行拍照和计数;
结果如图12和图13所示,RNA piR-hsa-164586构建的激动剂能够显著促进非小细胞肺癌细胞的迁移能力,RNA piR-hsa-164586 构建的抑制剂能够显著抑制非小细胞肺癌细胞的迁移能力。
实施例14:
本实施例涉及划痕实验对非小细胞肺癌细胞A549和HCC2279迁移能力的分析过程:
在六孔板中培养非小细胞肺癌细胞,对实验组进行RNA piR- hsa-164586的转染,转染两天后,用200ul移液器吸头刮擦细胞单层,通过在划擦后0h和24h拍摄10x高倍视野捕获细胞迁移的代表性图像,测量跨越诱导损伤区域的缩小距离,并将其标准化为0h对照,表示为相对迁移速率;
结果如图14和图15所示,RNA piR-hsa-164586构建的激动剂能够显著促进非小细胞肺癌细胞的迁移能力,RNA piR-hsa-164586 构建的抑制剂能够显著抑制非小细胞肺癌细胞的迁移能力。
实施例15:
本实施例涉及流式细胞仪对非小细胞癌细胞A549和HCC2279凋亡的检测过程:
培养非小细胞肺癌细胞,对实验组进行RNA piR-hsa-164586的转染,转染两天后,进行Annexin V-FITC/PI(膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶)细胞凋亡检测(美仑,MA0220):
(1)用不含EDTA(乙二胺四乙酸)的胰酶消化非小细胞肺癌细胞后,在300g,4℃的条件下离心5min,收集细胞;
(2)用预冷的PBS洗涤细胞2次,每次均需在300g,4℃的条件下离心5min,收集1-5×105细胞;
(3)吸弃PBS,加入1×Binding Buffer(染色缓冲液)重悬细胞;
(4)加入Annexin V-FITC(膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶)和PIStaining Solution(碘化丙啶染色液),轻轻混匀,于避光、室温条件下反应10-15分钟;
(5)加入1×Binding Buffer,混匀后置于冰上,在1h内用流式细胞仪检测;
结果如图16所示,RNA piR-hsa-164586构建的激动剂能够明显减少非小细胞肺癌细胞A549和HCC2279的凋亡,RNA piR-hsa- 164586构建的抑制剂能够明显增加非小细肺癌细胞A549和HCC2279 的凋亡。
Claims (2)
1.一种检测血清外泌体来源的piR-hsa-164586的引物在制备非小细胞肺癌检测效能评估试剂中的应用,其特征在于,piR-hsa-164586的核苷酸序列片段为TGAGAACTGAATTCCATAGGCTGC,引物为Human-piR-hsa-164586,上游引物序列Human-piR-hsa-164586-F为CGCGTGAGAACTGAATTCCATA,下游引物序列Human-piR-hsa-164586-R为AGTGCAGGGTCCGAGGTATT。
2.一种检测血清外泌体来源的piR-hsa-164586的引物在制备非小细胞肺癌组织术后预后评估试剂中的应用,其特征在于,piR-hsa-164586的核苷酸序列片段为TGAGAACTGAATTCCATAGGCTGC,引物为Human-piR-hsa-164586,上游引物序列Human-piR-hsa-164586-F为CGCGTGAGAACTGAATTCCATA,下游引物序列Human-piR-hsa-164586-R为AGTGCAGGGTCCGAGGTATT。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210628527.3A CN114959033B (zh) | 2022-06-06 | 2022-06-06 | 一种外泌体piRNA的用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210628527.3A CN114959033B (zh) | 2022-06-06 | 2022-06-06 | 一种外泌体piRNA的用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114959033A CN114959033A (zh) | 2022-08-30 |
CN114959033B true CN114959033B (zh) | 2024-05-14 |
Family
ID=82959804
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210628527.3A Active CN114959033B (zh) | 2022-06-06 | 2022-06-06 | 一种外泌体piRNA的用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114959033B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN118109582A (zh) * | 2022-11-29 | 2024-05-31 | 四川大学华西医院 | 检测piR-hsa-790910的试剂在制备肺癌筛查或诊断试剂盒中的用途 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112402627A (zh) * | 2020-11-18 | 2021-02-26 | 青岛大学 | 一种PIWI蛋白相互作用RNA piR-hsa-211106的用途 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20200017906A1 (en) * | 2018-02-12 | 2020-01-16 | Sun Yat-Sen University | Use of pirna-54265 in diagnosis, treatment, and prognostic evaluation of colorectal cancer |
-
2022
- 2022-06-06 CN CN202210628527.3A patent/CN114959033B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112402627A (zh) * | 2020-11-18 | 2021-02-26 | 青岛大学 | 一种PIWI蛋白相互作用RNA piR-hsa-211106的用途 |
Also Published As
Publication number | Publication date |
---|---|
CN114959033A (zh) | 2022-08-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2020200114B2 (en) | Methods and assays relating to circulating tumor cells | |
CN108315414B (zh) | 一种预测食管鳞状细胞癌预后的生物标志物 | |
CN114959033B (zh) | 一种外泌体piRNA的用途 | |
CN106834486B (zh) | 骨肉瘤分子诊疗标志物及其应用 | |
CN111172283B (zh) | 检测linc00673表达量在食管癌靶向治疗预后评估试剂盒中的应用 | |
CN112961913A (zh) | lncRNA在复发性流产诊治中的应用 | |
CN109825587B (zh) | 一种胶质瘤预后标志物cpvl及其应用 | |
CN105483275A (zh) | mir-1299及其成熟miRNA的新用途 | |
CN110878350B (zh) | 一种用于脓毒症诊断及预后评估的试剂盒 | |
CN110205386B (zh) | 一种与子宫内膜癌相关的miRNA分子miR-33a-5p及其应用 | |
CN112114143A (zh) | 一种肝癌诊断及治疗致癌激酶标志物的应用 | |
CN111394465A (zh) | 结直肠癌奥沙利铂耐药相关的lncRNA的筛选及应用 | |
CN116577506A (zh) | 一种鉴别hcc亚型的标志物hnf1b及dphcc体外模型的构建 | |
CN111979315A (zh) | 环状tp63作为肺鳞癌诊断或治疗靶点的应用 | |
CN114487419A (zh) | 一种tjp3基因编码蛋白在制备卵巢癌检测试剂中的应用 | |
CN105505936A (zh) | 一种抗骨肉瘤转移生物制剂及其应用 | |
CN113403396B (zh) | 针对Sorcin调控环路诊断、预测、逆转卵巢癌紫杉醇耐药 | |
CN115896263B (zh) | LncRNA ENST00000440246.1在阿尔茨海默病中的应用 | |
US20230250425A1 (en) | USE OF PIWI-INTERACTING RNA piR-hsa-211106 | |
Yang et al. | Effects of MiR-194-5p on the proliferation and invasion of laryngeal cancer cells by regulating the expression of smurf1 and activating the mTOR signaling pathway | |
CN113652487B (zh) | 人C11orf86 mRNA在评估肺腺癌患者预后中的应用及检测试剂盒 | |
CN113403390B (zh) | lncRNA在儿童心肌炎诊治中的应用 | |
CN105597109A (zh) | 原发骨肉瘤的诊治分子标记 | |
CN116716401A (zh) | 生物标志物polq在制备检测或评估胶质瘤预后情况的产品中的应用 | |
CN112986559A (zh) | H基因及其表达产物在鉴定、筛选或分选肝癌干细胞中的应用 |
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 |