CN116769919A - Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof - Google Patents
Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof Download PDFInfo
- Publication number
- CN116769919A CN116769919A CN202310912767.0A CN202310912767A CN116769919A CN 116769919 A CN116769919 A CN 116769919A CN 202310912767 A CN202310912767 A CN 202310912767A CN 116769919 A CN116769919 A CN 116769919A
- Authority
- CN
- China
- Prior art keywords
- ovarian cancer
- gene
- tumor cell
- metastasis
- molecular marker
- 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.)
- Pending
Links
- 206010033128 Ovarian cancer Diseases 0.000 title claims abstract description 105
- 206010061535 Ovarian neoplasm Diseases 0.000 title claims abstract description 105
- 210000004881 tumor cell Anatomy 0.000 title claims abstract description 60
- 239000000203 mixture Substances 0.000 title claims abstract description 44
- 206010027476 Metastases Diseases 0.000 title claims abstract description 43
- 230000009401 metastasis Effects 0.000 title claims abstract description 43
- 239000003147 molecular marker Substances 0.000 title claims abstract description 39
- 230000001737 promoting effect Effects 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 91
- 108010052495 Calgranulin B Proteins 0.000 claims abstract description 28
- 102100032420 Protein S100-A9 Human genes 0.000 claims abstract description 28
- 101000972834 Homo sapiens Normal mucosa of esophagus-specific gene 1 protein Proteins 0.000 claims abstract description 19
- 102100026443 Adhesion G-protein coupled receptor F1 Human genes 0.000 claims abstract description 18
- 108010052500 Calgranulin A Proteins 0.000 claims abstract description 18
- 102100025473 Carcinoembryonic antigen-related cell adhesion molecule 6 Human genes 0.000 claims abstract description 18
- 102100026891 Cystatin-B Human genes 0.000 claims abstract description 18
- 101000718228 Homo sapiens Adhesion G-protein coupled receptor F1 Proteins 0.000 claims abstract description 18
- 101000914326 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 6 Proteins 0.000 claims abstract description 18
- 101000912191 Homo sapiens Cystatin-B Proteins 0.000 claims abstract description 18
- 101000884770 Homo sapiens Cystatin-M Proteins 0.000 claims abstract description 18
- 101000972286 Homo sapiens Mucin-4 Proteins 0.000 claims abstract description 18
- 101000995300 Homo sapiens Protein NDRG2 Proteins 0.000 claims abstract description 18
- 101000874179 Homo sapiens Syndecan-1 Proteins 0.000 claims abstract description 18
- 102100022693 Mucin-4 Human genes 0.000 claims abstract description 18
- 102100034436 Protein NDRG2 Human genes 0.000 claims abstract description 18
- 102100032442 Protein S100-A8 Human genes 0.000 claims abstract description 18
- 102100035721 Syndecan-1 Human genes 0.000 claims abstract description 18
- -1 PI3 Proteins 0.000 claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 14
- 201000011510 cancer Diseases 0.000 claims abstract description 10
- 230000035755 proliferation Effects 0.000 claims abstract description 10
- 239000003112 inhibitor Substances 0.000 claims abstract description 9
- 238000001727 in vivo Methods 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims description 64
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 108020004459 Small interfering RNA Proteins 0.000 claims description 18
- 239000002924 silencing RNA Substances 0.000 claims description 13
- 102100022646 Normal mucosa of esophagus-specific gene 1 protein Human genes 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 108020004999 messenger RNA Proteins 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 238000003753 real-time PCR Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 101150096430 CEACAM6 gene Proteins 0.000 claims description 2
- 101150023402 CST6 gene Proteins 0.000 claims description 2
- 101100108384 Homo sapiens ADGRF1 gene Proteins 0.000 claims description 2
- 101150059949 MUC4 gene Proteins 0.000 claims description 2
- 101150096352 Ndrg2 gene Proteins 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 101150032327 PI3 gene Proteins 0.000 claims description 2
- 101150060340 S100a8 gene Proteins 0.000 claims description 2
- 101150012953 S100a9 gene Proteins 0.000 claims description 2
- 101150002135 SDC1 gene Proteins 0.000 claims description 2
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 2
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 2
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000007899 nucleic acid hybridization Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000004055 small Interfering RNA Substances 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000013518 transcription Methods 0.000 claims description 2
- 230000035897 transcription Effects 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims 1
- 230000002452 interceptive effect Effects 0.000 claims 1
- 230000001394 metastastic effect Effects 0.000 abstract description 12
- 206010061289 metastatic neoplasm Diseases 0.000 abstract description 12
- 238000011065 in-situ storage Methods 0.000 abstract description 8
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 238000004393 prognosis Methods 0.000 abstract description 4
- 230000002596 correlated effect Effects 0.000 abstract description 3
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 238000002651 drug therapy Methods 0.000 abstract 1
- 238000001415 gene therapy Methods 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 31
- 210000001519 tissue Anatomy 0.000 description 17
- 241000699660 Mus musculus Species 0.000 description 15
- 238000011580 nude mouse model Methods 0.000 description 15
- 238000010790 dilution Methods 0.000 description 13
- 239000012895 dilution Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 238000007920 subcutaneous administration Methods 0.000 description 12
- 230000002401 inhibitory effect Effects 0.000 description 10
- 239000003550 marker Substances 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 9
- 238000002054 transplantation Methods 0.000 description 8
- 238000011532 immunohistochemical staining Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 230000001575 pathological effect Effects 0.000 description 6
- 238000009826 distribution Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000005012 migration Effects 0.000 description 5
- 238000013508 migration Methods 0.000 description 5
- 238000010837 poor prognosis Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 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
- 210000002216 heart Anatomy 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000009545 invasion Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 102000013127 Vimentin Human genes 0.000 description 3
- 108010065472 Vimentin Proteins 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108020005196 Mitochondrial DNA Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012174 single-cell RNA sequencing Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 210000005048 vimentin Anatomy 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 238000007808 Cell invasion assay Methods 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102100034836 Proliferation marker protein Ki-67 Human genes 0.000 description 1
- 101710194663 Proliferation marker protein Ki-67 Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 101150036080 at gene Proteins 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 230000003021 clonogenic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 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
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004547 gene signature Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000000513 principal component analysis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 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 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 238000011870 unpaired t-test Methods 0.000 description 1
- 230000003442 weekly effect Effects 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- 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 N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- 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
- C12Q2600/136—Screening for pharmacological compounds
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Pathology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Hospice & Palliative Care (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The application discloses a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof, and the application discovers that a group of tumor cell subgroups taking expressed S100A9 as a main characteristic exist in-situ tissues and metastatic tissues of ovarian cancer, and the tumor cell subgroup is positively correlated with the bad prognosis of ovarian cancer patients, and identifies the specific tumor cell molecular marker composition for promoting ovarian cancer metastasis: the S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 genes are all expressed in a specific and significant manner. Inhibition of these molecular markers can significantly inhibit proliferation and metastasis of ovarian cancer cells in vitro, as well as malignant growth in vivo. The inhibitor can be used for preparing a pharmaceutical composition for preventing or treating ovarian cancer metastasis, and can be used for clinical applications such as gene therapy, drug therapy and the like of ovarian cancer. In addition, the kit provided by the application is used for auxiliary diagnosis and prognosis prediction analysis of ovarian cancer metastasis, and has important guiding significance for subsequent clinical treatment.
Description
Technical Field
The application relates to the technical field of tumor molecular biology, in particular to a tumor cell subset with S100A9 expression as a main characteristic, and further relates to application of a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis.
Background
Ovarian cancer is one of the common malignant tumors of female reproductive organs, and the mortality rate is high at the top. According to the global cancer burden data issued by the international cancer research institute of the world health organization in 2021, the new occurrence of the global ovarian cancer is about 31 ten thousand, and the death cases are over 20 ten thousand, so that the life and the health of women are seriously threatened. Because the incidence of ovarian cancer is hidden, more than 70% of patients are found to be in late stage and abdominal cavity multi-site metastasis occurs more often, thus greatly impeding the feasibility and safety of the operation. The current treatment means is unsatisfactory, the survival rate of patients in the advanced stage for 5 years is always less than 40%, but the survival rate of patients without combined transfer for 5 years can reach more than 90%. The easy occurrence and metastasis of the ovarian cancer are important reasons for restricting the survival rate of patients with the ovarian cancer to be improved, and are also difficult treatment problems faced by clinicians. Thus, effective suppression of metastasis of ovarian cancer would help improve the poor prognosis of ovarian cancer patients.
The single-cell sequencing technology is a current hot spot research means, has obvious advantages compared with the traditional sequencing technology in the aspects of exploring tumor cell heterogeneity and cancer cell evolution track, analyzing complex tumor microenvironment, analyzing the interaction of tumor cells with tumor microenvironment cells such as interstitial cells, immune cells and the like, and is one of the major scientific breakthroughs of 2018 year 10. However, studies on ovarian cancer metastasis based on single cell sequencing remain to be explored further. Therefore, the patent study carries out deep single-cell transcriptome sequencing on tumor primary sites of 18 ovarian cancer primary patients and/or tumor tissues (34 samples in total) of different metastasis sites, and establishes high-resolution ovarian cancer in-situ and metastasis cell maps. The basic exploration of the ovarian cancer is assisted by utilizing a single cell sequencing technology, and the molecular characteristics of ovarian cancer metastasis are combined, so that the key tumor cell population in the disease evolution process is explored, the accurate targeted treatment is hopeful, and finally, the survival ending of an ovarian cancer patient is improved.
Disclosure of Invention
The application aims at overcoming the defects of the prior art and provides a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof.
The aim of the application is realized by the following technical scheme:
single cell transcriptome sequencing was performed on tumor tissue (34 samples total) taken from tumor primary sites and/or different metastasis sites of 18 ovarian cancer primary patients, and high resolution ovarian cancer in situ and metastasis maps were established. A small population of cell subsets characterized by expression of S100A9 was found in the map in situ tumor cells. The tumor cell subpopulation is positively correlated with a poor prognosis for ovarian cancer. The tumor cell subpopulations exist in different patients, not individual-specific, and their presence is common. The tumor cell subpopulation is also prevalent in tumor cells at the metastatic site of ovarian cancer. Through further analysis of the common molecular expression characteristics of S100deg.A < 9+ > tumor cells existing in-situ tumor tissues and metastatic tissues, a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis is found, and the specific tumor cell molecular marker composition is remarkably and highly expressed. The specific tumor cell molecular marker composition is a characteristic molecule with obviously high expression specificity shared by tumor cell subsets with S100A9 as a main characteristic, and the molecule or the combination thereof is selected from any one or more of (a) - (j): (a) the S100A8 gene and its expression product; (b) the S100A9 gene and its expression product; (c) an ADGRF1 gene and its expression product; (d) CEACAM6 gene and its expression product; (e) CST6 gene and its expression product; (f) NDRG2 gene and its expression product; (g) MUC4 gene and its expression product; (h) PI3 gene and its expression product; (i) SDC1 gene and its expression product; (j) C15orf48 gene and its expression product.
Further, the gene ID encoding S100A8, NCBI database, is the sequence indicated by 6279;
the gene ID of NCBI database coding S100A9 is 6280;
the gene ID of NCBI database encoding ADGRF1 is 266977;
the gene ID of NCBI database coding CEACAM6 is 4680;
the gene ID of NCBI database encoding CST6 is 1474;
the gene ID of the NCBI database coding NDRG2 is a sequence shown as 57447;
the gene ID encoding MUC4, NCBI database, is the sequence shown as 4585;
the gene ID of NCBI database encoding PI3 is 5266;
the gene ID of NCBI database encoding SDC1 is 6382;
the gene ID of NCBI database encoding C15orf48 is 84419;
the S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 genes and their expression products are all human sources.
Application of a specific tumor cell molecular marker composition reagent for identifying or detecting ovarian cancer metastasis promotion in preparation of medicines for diagnosing, detecting, monitoring or predicting the progress of ovarian cancer.
Further, the progression of ovarian cancer includes proliferation and metastasis of ovarian cancer cells in vitro, as well as malignant growth in vivo. The diagnosis includes: whether an ovarian cancer is a poor prognosis, wherein high expression is a poor prognosis of ovarian cancer; detecting, monitoring, or predicting the progression of ovarian cancer includes: detecting the expression of the molecular marker composition in the patient, wherein the high expression is proliferation and metastasis of ovarian cancer cells in vitro and the likelihood of malignant growth in vivo is greater.
Application of a reagent for inhibiting expression of a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis in preparing medicines for treating or preventing ovarian cancer metastasis.
A kit for diagnosing, detecting, monitoring or predicting the progression of ovarian cancer, an agent that identifies or detects a marker molecule selected from the group consisting of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48, or any combination thereof;
the progression of ovarian cancer includes proliferation and metastasis of ovarian cancer cells in vitro, as well as malignant growth in vivo.
Further, mRNA of a marker molecule or cDNA of the marker molecule is detected by a nucleic acid detection method; preferably, transcription analysis, real-time fluorescent quantitative PCR, nucleic acid hybridization or any combination thereof is employed. The reagents each independently comprise at least one primer or probe complementary to a portion of the nucleotide sequence of an mRNA or cDNA of the specific tumor cell molecular marker composition of the type that promotes metastasis of ovarian cancer. The kit at least comprises one or more of the following 10 primer pairs, and the primer sequences of the real-time quantitative reverse transcription PCR are shown in SEQ ID NO. 3-22.
A pharmaceutical composition for treating or preventing metastasis of ovarian cancer, the pharmaceutical comprising a substance that inhibits the S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and/or C15orf48 genes and expression products and/or reduced activity thereof, or any combination thereof.
Further, the inhibitor at least comprises a protein-specific antibody, an RNA interference molecule or antisense oligonucleotide aiming at gene mRNA, a small molecule inhibitor, siRNA and shRNA.
Further, the design of specific siRNAs for each of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 was used, and the siRNA sequences were as shown in SEQ ID NO.23-SEQ ID NO. 62. Or a construct capable of expressing or forming said siRNA.
In vitro functional experiments show that in OVCAR3 and CAOV3 ovarian cancer cells, inhibition of the expression of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 or C15orf48 can significantly inhibit the proliferation activity and the metastasis capability of the ovarian cancer cells.
In vivo OVCAR3 nude mice subcutaneous transplantation tumor experiments show that inhibiting the expression of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 or C15orf48 can obviously inhibit the growth capacity and metastasis related protein expression of nude mice subcutaneous transplantation tumor. The tumorigenic effects of S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 in ovarian cancer cells were initially determined.
The beneficial effects of the application are as follows: the application separates out tumor cell subgroup with different functions through single cell group analysis, and identifies specific marker molecules of the subgroup. The application provides a specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof. The molecular marker composition has the capability of obviously inhibiting proliferation and metastasis of ovarian cancer cells in vitro and malignant growth in vivo. The results show that the molecular marker composition is an important cancerogenic factor in ovarian cancer, can be used as an important index for diagnosing, detecting, monitoring or predicting the progress of ovarian cancer, and can be used as a therapeutic target and a prognostic index of ovarian cancer metastasis.
Drawings
Fig. 1: unsupervised cluster map of heterogeneous subpopulations present in ovarian cancer tissue. UMAP plots show that 22 different color-divided cell clusters are shown by differences in gene expression of the cells.
Fig. 2: schematic representation of major cell types in ovarian cancer in situ tissue and metastatic tissue. (A) UMAP plots show the major cell types in ovarian cancer in situ tissue and metastatic tissue. (B) The dot plot shows the expression level of a particular marker gene in each cell type. The size of the dot indicates the proportion of cells expressing a particular marker gene. The color spectrum represents the average expression level of the marker gene.
Fig. 3: unsupervised cluster map of heterogeneous subpopulations of tumor cells present in ovarian cancer tissue. (A) Ovarian cancer tumor cells are divided into six subgroups according to the gene expression difference and molecular characteristics of the tumor cells. (B) The survival analysis curve of the S100A9+ tumor cell subset shows that ovarian cancer patients with high expression of the S100A9 tumor cell subset (S100A 9-tumor cells are S100A9 negative tumor cells) have poorer prognosis. The Kaplan-Meier survival curve showed significant prognostic isolation. Analysis of total 5 year survival time (OS) from 373 patients with primary ovarian cancer based on the s100deg.A9+ tumor cell marker gene signature from single cell data.
Fig. 4: distribution of tumor cell subsets in individual patients with ovarian cancer. The s100deg.A 9+ tumor cell subpopulation is present in different patients, not individual-specific, and its presence is common.
Fig. 5: specific gene expression levels in each tumor cell subpopulation. The dot plot shows the expression level of the specific marker gene in each tumor cell subpopulation. The S100A9+ tumor cell subsets share the molecular expression characteristics, and S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 are all expressed with high specificity.
Fig. 6: UMAP schematic representation of 10 heterogeneous subpopulations of tumor cells present in ovarian cancer metastasis tissue. A subset of s100deg.A 9+ tumor cells are also present in ovarian cancer metastasis tumor cells.
Fig. 7: distribution of tumor cell subsets in metastatic tissues of ovarian cancer at each group of metastatic sites. The s100deg.A 9+ tumor cell subsets were present in different metastatic tissues.
Fig. 8: designing schematic diagrams of knockdown efficiency results of two pairs of siRNAs in ovarian cancer cells for each gene respectively; (A) Knockdown efficiency results for each pair of siRNAs in human ovarian cancer cell line OVCAR 3. (B) Knockdown efficiency results for each pair of siRNAs in the human ovarian cancer cell line CAOV 3.
Fig. 9: results of inhibiting the effect of expression of specific molecular marker compositions on the proliferative capacity of human ovarian cancer cell line OVCAR3 schematic (a) and statistical graph (B)
Fig. 10: results of inhibition of expression of specific molecular marker compositions on proliferation potency of human ovarian cancer cell line CAOV3 schematic (A) and statistical (B)
Fig. 11: results of inhibiting the effect of expression of a specific molecular marker composition on the migration ability of human ovarian cancer cell line OVCAR3 schematic (a) and statistical graph (B).
Fig. 12: results of inhibiting the effect of expression of a specific molecular marker composition on the invasive capacity of the human ovarian cancer cell line OVCAR3 schematic (a) and statistical graph (B).
Fig. 13: results of inhibition of expression of specific molecular marker compositions on the ability of the human ovarian cancer cell line CAOV3 to migrate are shown schematically (a) and statistically (B).
Fig. 14: results of inhibition of expression of specific molecular marker compositions on the invasive capacity of the human ovarian cancer cell line CAOV3 are shown in schematic (a) and statistical (B).
Fig. 15: human ovarian cancer cell line OVCAR3 nude mice subcutaneous engrafting tumor model different groups of engrafting tumors were photographed by digital cameras (a) and growth curves of nude mice subcutaneous engrafting tumors (B).
Fig. 16: immunohistochemical staining schematic (a) and statistical (B) of the pathological section cell proliferation marker protein Ki67 of different groups of human ovarian cancer cell line OVCAR3 nude mice subcutaneous engrafting tumor model.
Fig. 17: immunohistochemical staining schematic (A) and statistical chart (B) of different groups of pathological section cell transfer related protein Vimentin of human ovarian cancer cell line OVCAR3 nude mice subcutaneous transplantation tumor model.
Fig. 18: immunohistochemical images of different groups of pathological sections of human ovarian cancer cell line OVCAR3 nude mice subcutaneous engrafting tumor model. (A) Immunohistochemical staining of different groups of pathological sections was performed with antibodies to S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 or C15orf48, respectively. (B) Different groups of pathological sections were subjected to immunohistochemical staining statistics with antibodies to S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 or C15orf48, respectively.
Fig. 19: in vivo OVCAR3 model H & E pathological section images of major organs (heart, liver, spleen, lung, kidney) collected from different treatment groups.
Detailed Description
The application will be further described with reference to examples and figures
Example 1: single cell sequencing analysis found s100deg.A 9+ tumor cell subpopulation, illustrating its characteristics and specific molecular expression profile 1. Single cell isolation: ovarian cancer tissue and metastatic tissue samples were surgically excised and minced into small pieces (about 1 mm) on ice 3 Size). Subsequently, collagenase I (Sigma), collagen was usedEnzyme IV (Sigma) and DNase I (Worthington) were digested in a water bath at 37℃for 30 minutes. After digestion, the samples were passed through a 40 μm cell filter and centrifuged at 300 Xg for 5 minutes. For ascites, cells in the ascites were directly filtered through a 40 μm cell filter and then centrifuged at 300×g for 5 minutes. The supernatant was discarded and the pellet resuspended in erythrocyte lysis buffer (Solarbio) to remove erythrocytes. The cells were then resuspended in RPMI1640 complete medium and filtered through a 35 μm cell filter. Isolated single cells were stained with acridine orange/propidium iodide (AO/PI) and cell viability was assessed using a Countstar fluorescent cell analyzer. Single cell RNA sequencing was then performed using 10X Genomics Chromium Controller Instrument to collect single cell suspensions and analyzed as follows:
the scRNA-seq data is filtered out of the adapter sequence using default parameters, removing low quality reads, and obtaining clean data. The reads were then compared to the human genome (GRCh 38 ensembl: v 91) to generate a characteristic barcode matrix. Based on the barcode reading of each cell of each sample, a downsampling analysis is performed on the sequenced samples and an aggregation matrix is generated as a final output. Cells expressing more than 200 genes and having a mitochondrial UMI rate below 20% pass the quality filter criteria, mitochondrial genes are excluded from the expression table.
Cell normalization and regression were then performed according to the expression table, while taking into account UMI counts and percentage of mitochondrial genes for each sample, resulting in scaled data. And carrying out principal component analysis on the scale data by using the first 2000 high-variable genes, and selecting the first 10 principal components for Uniform Manifold Approximation and Projection (UMAP) construction. By adopting the graph-based clustering method, unsupervised cell clustering is obtained based on the first 10 principal components of PCA. Determining the marker genes of each cluster using the criteria of 1) log values of fold differences (lnFC) > 0.25; 2) Assumed value < 0.05; 3) Minimum percentage (min. Pct) > 0.1. To further determine a particular cell type, clusters representing the same cell type are selected for subsequent re-UMAP analysis, graph-based clustering, and marker analysis.
2. Survival analysis: the total survival of ovarian cancer patients was studied from a large amount of mRNA-seq data obtained from the cancer genomic profile (TCGA) database.
3. Results: the high resolution cell patterns of tumor primary and/or metastatic sites of 18 patients with ovarian cancer initiation are shown in fig. 1-2, and as shown in fig. 3, a small group of cell subsets mainly characterized by expressing S100A9 exist in-situ tumor cells (fig. 3A), and TCGA ovarian cancer database prognosis analysis shows that s100deg.A9+ tumor cell subsets are positively correlated with the poor prognosis of ovarian cancer (fig. 3B). As shown in fig. 4, the s100deg.A 9+ tumor cell subpopulations were present in different patients, not individual-specific, and their presence was common. As shown in FIG. 5, a class of specific tumor cell molecular marker compositions for promoting ovarian cancer metastasis is identified by further analyzing the expression characteristics of S100deg.A 9+ tumor cell common molecules: S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 genes, and are all remarkably high in specificity. s100deg.A 9+ tumor cells were also present in tumor cells at the metastatic sites of ovarian cancer (FIG. 6), and were present at different metastatic sites (FIG. 7).
Example 2 Effect of inhibitors of molecular marker compositions on proliferation and invasion of ovarian cancer cells
1. Conventional cell culture: human ovarian cancer cell line CAOV3 was cultured in DMEM medium containing 10vol% FBS, human ovarian cancer cell line OVCAR3 was cultured in RPMI-1640 medium containing 10vol% FBS and placed in 5vol% CO 2 Conventional culture in a 37 ℃ cell incubator, liquid exchange every 2-3 days, passage every time the cell fusion degree reaches 90%, and taking the cells in the logarithmic growth phase for subsequent experiments.
2. Cell transfection:
1) siRNA synthesis: and selecting action targets for different molecules, designing according to the principle of determining sequences, and synthesizing siRNA for different molecules.
Table 1 the sequences of siRNA.
2) The CAOV3 or OVCAR3 cells were seeded at a density of 50 ten thousand per well in six well plates and transfected when the cells had fused to about 50% -60% of the adherent growth. Taking 1-well liquid addition amount of a six-well plate as an example:
and (3) solution A: 2.5. Mu.L of siRNA and 97.5. Mu.L of OPTI-MEM were mixed;
and (2) liquid B: 2.5. Mu.L of DharmaFECT and 97.5. Mu.L of OPTI-MEM were mixed;
and (3) after standing at room temperature for 5min, uniformly mixing the solution A and the solution B, standing at room temperature for 20min, preparing a working solution, and then uniformly mixing without vigorous blowing. After adding 0.8ml of the medium to the six-well plate, 200. Mu.L of the above working solution was slowly added dropwise so as not to cause too much stimulation to cells. CO at 37 DEG C 2 After 24 hours of incubation in the incubator, 2ml of complete medium was changed per well, and the knockdown efficiency was detected at 48 hours, and the subsequent functional experiments.
Rna extraction: six well plates were added with 1mL Trizol per well, allowed to stand at room temperature for 15 minutes for lysis, and transferred to RNase-free centrifuge tubes. 200. Mu.L of chloroform was added to each 1mL of Trizol, and the mixture was mixed upside down for 15s, left at room temperature for 15 minutes, and centrifuged at 14000rpm at 4℃for 15 minutes. After centrifugation, the liquid is divided into three layers, wherein the upper layer is a colorless transparent layer, namely RNA; the bottom layer is red organic phase, and the middle flocculent precipitate layer is mainly DNA. The upper aqueous phase was aspirated into another clean centrifuge tube, taking care not to aspirate the middle pellet. Add 500. Mu.L of isopropanol to the aspirated supernatant, mix upside down for 15s, incubate at room temperature for 15 minutes, centrifuge at 12000rpm at 4℃for 15 minutes. After centrifugation, a white precipitate was evident at the bottom of the tube, the supernatant was discarded, and the RNA pellet was washed twice with 1mL of pre-chilled 75vol% ethanol and centrifuged at 7500rpm for 5 minutes at 4 ℃. The EP tube was inverted and dried on clean absorbent paper, and 10. Mu.L-50. Mu.L DEPC water was added to dissolve the RNA well depending on the size of the pellet. 1 mu L of RNA solution is taken to detect the concentration and purity of RNA by a micro nucleic acid quantitative analyzer, and the OD260/OD280 of the RNA with better quality is between 1.8 and 2.2. Preserving in a refrigerator at-80deg.C.
4. Reverse transcription and qPCR: reverse transcription was performed using ABScript Neo RT Master Mix for qPCR with gDNA Remover (abclon, RK 20433). Amplification reactions were performed using 2X Universal SYBR Green Fast qPCR Mix (ABclonal, RK 21203) on a Light cycle 480 instrument (Roche). The primer sequences of specific targets are shown in Table 2. Obtaining CT value of each sample according to 2 -ΔΔCT The method calculates the relative expression of the target gene.
Table 2: RT-PCR detection fragment primer sequence
5. Cell clone proliferation assay: each group had a cell density of 1 x 10 at 48 hours post transfection 3 Well, inoculated in 6-well plate. After 14 days of incubation at 37℃the cells were washed, fixed and stained with 0.5wt% crystal violet dissolved in 20vol% methanol. Cell colony formation per well was counted.
6. Statistical analysis: statistical analysis was performed using Graphpad Prism 8.0.1 software. Results are expressed as mean±sd. Data with normal distribution and uneven variance were compared using Student's t, and data with normal distribution and uneven variance were compared using Welch's correction unpaired t-test. Non-normal distribution experimental data was tested using the non-parametric Mann-Whitney test. P <0.05 is statistically significant for the differences.
7. Results: siRNA transfection efficiency was examined and the results are shown in FIG. 8 to be statistically significant in terms of knockdown efficiency in both ovarian cancer cells OVCAR3 (FIG. 8A) and CAOV3 (FIG. 8B). After knocking down the expression of the corresponding molecules, the clonogenic capacity of both ovarian cancer cells OVCAR3 (fig. 9) and CAOV3 (fig. 10) was significantly reduced. It is shown that inhibiting any molecular expression of the molecular marker composition can significantly inhibit ovarian cancer cell proliferation.
Example 3 effect of inhibitors of molecular marker compositions on migration and invasion of ovarian cancer cells.
Tranwell cell invasion assay: spreading glue: BD matrigel was boiled on ice in advance, and a 1.5ml sterile EP tube, a tip box with 200. Mu.L tips, and a Transwell plate were placed in a refrigerator for pre-cooling. Mixing BD matrigel and precooled OPTI-MEM at a volume ratio of 1:10, taking 100 μL, adding into an upper chamber in a Transwell plate, taking out the Transwell plate after slightly placing into an incubator for standing for 30min, carefully sucking the upper layer non-coagulated liquid, and coating BD gel. Cells were harvested by digestion 48h after cell transfection and resuspended in serum-free medium; 20 ten thousand cells were resuspended in 200 μl of serum-free medium in the upper chamber; the lower chamber was placed with a high concentration medium containing 20vol% FBS. CAOV3 cells were incubated in an incubator at 37℃for 24h, after incubation of OVCAR3 cells for 12h, 0.5wt% crystal violet was fixed and stained for 20min, gently rinsed with clear water, and the upper cells were carefully wiped off with a cotton swab, photographed and counted.
2. Migration experiment: cells were harvested by digestion 48h after cell transfection and resuspended in serum-free medium; 10 ten thousand cells were resuspended in 200 μl of serum-free medium in the upper chamber; the lower chamber was placed with a high concentration medium containing 20vol% FBS. CAOV3 cells were incubated in an incubator at 37℃for 24h, after incubation of OVCAR3 cells for 12h, 0.5wt% crystal violet was fixed and stained for 20min, gently rinsed with clear water, and the upper cells were carefully wiped off with a cotton swab, photographed and counted.
3. The results are shown in fig. 11-12, which demonstrate that inhibiting any molecular expression of the molecular marker composition can significantly inhibit the migration (fig. 11) and invasion (fig. 12) ability of ovarian cancer cells OVCAR3, and as such, inhibiting any molecular expression of the molecular marker composition can significantly inhibit the migration (fig. 13) and invasion (fig. 14) ability of ovarian cancer cells CAOV 3.
Example 4 Effect of inhibitors of molecular marker compositions on the growth of ovarian cancer cell nude mice subcutaneous transplantations
1. Establishment of nude mice subcutaneous transplantation tumor: OVCAR3 cells were transfected with different siRNAs (si-NC, si-S100A8, si-S100A9, si-ADGRF1, si-CEACAM6, si-CST6, si-NDRG2, si-MUC4, si-PI3, si-SDC1 and si-C15orf 48), centrifuged after 24h and washed twice with PBS to wash out serum. Resuspension counts of 1 x 10 per nude mice per group of 4 mice 7 Cells were subcutaneously injected under the axilla of female nude mice (4 week old, BALB/c nude mice). Tumor formation was observed 1 week after injection of cells, and tumor volume (V) was monitored weekly by measuring long diameter (L) and wide diameter (W) of the tumor seeds every other week and calculated using formula v=0.5×l×w×w. After 14 days, 1nmol of siRNA (si-NC, si-S100A8, si-S100A9, si-ADGRF1, si-CEACAM6, si-CST6, si-NDRG2, si-MUC 4),si-PI3, si-SDC1, and si-C15orf 48). Mice were euthanized after 28 days, tumors were removed, their volumes were measured and the volume and weight sizes of tumors were weighed against the control and experimental groups. Collecting viscera (heart, liver, spleen, lung, and kidney), soaking tumor tissue and viscera in 4vol% paraformaldehyde solution for subsequent hematoxylin and eosin (H)&E) Staining and immunohistochemical staining. Animal experiments were performed according to guidelines approved by the committee for laboratory animal management and ethics.
2. Hematoxylin and eosin staining and immunohistochemical staining: paraffin embedding after tissue sample fixation. Hematoxylin and eosin (H & E) staining observed the histopathological features of each organ (heart, liver, spleen, lung, kidney). Immunohistochemical staining was performed on tumor sections. Sections were dewaxed, hydrated, permeabilized, blocked, then depurated with primary antibodies Ki-67 (Proteintech, 27309-1-AP,1:400 dilution), vimentin (Abcam, ab92547,1:400 dilution), S100A8 (Proteintech, 15792-1-AP,1:400 dilution), S100A9 (HUABIO, ET1702-73,1:200 dilution), ADGRF1 (Affinity Biosciences, DF2796,1:100 dilution), CEACAM6 (HUABIO, EM 1-68,1:500 dilution), CST6 (Proteintech, 17076-1-AP,1:200 dilution), NDRG2 (HUABIO, ER1802-94,1:100 dilution), MUC4 (HUABIO, ET1705-13,1:100 dilution), PI3 (Proteintech, 15961-AP, 1:100 dilution), SDC1 (HUIO, 1703-42,1:500 dilution) and Ab 190-1:200 dilution (Abcalif, 23:200 dilution). Sections were stained with hematoxylin to label the nuclei and observed under a microscope. For the Ki-67 analysis, image j was used to evaluate the percentage of positive cells. For other proteins, IHC scores were determined by multiplying the scores of the two components. First score: the percentage of positive cells was scored 0-25% 1,26-50% 2,51-75% 3,76-100% 4. Second score: the staining intensity was scored negative for 1, weak for 2, medium for 3, and strong for 4. At least 5 fields of view are analyzed per slice and the scores of the 5 fields of view are averaged to obtain a final score.
4. As shown in fig. 15, any of the molecular marker compositions inhibited expression significantly inhibited the growth ability of subcutaneous transplantation tumor in nude mice. As shown in fig. 16-17, any one of the molecular marker compositions can significantly inhibit the expression of ki67 (fig. 16) and the expression of vimentin (fig. 17) in the nude mice subcutaneous transplantation tumor, which indicates that any one of the molecular marker compositions can significantly inhibit the proliferation and the transfer capacity of the nude mice subcutaneous transplantation tumor. As shown in FIG. 18, any of the molecules using the siRNA inhibiting molecular markers had significant knockdown efficiency at the protein expression level. As shown in fig. 19, the use of siRNA inhibitors did not cause significant damage to important organs including heart, liver, spleen, lung and kidney of nude mice, and had good biosafety.
Claims (10)
1. A specific tumor cell molecular marker composition for promoting ovarian cancer metastasis, which is characterized by being a characteristic molecule with remarkably high expression specificity shared by tumor cell subsets with S100A9 as a main characteristic, wherein the molecule or the combination thereof is selected from any one or more of (a) - (j): (a) the S100A8 gene and its expression product; (b) the S100A9 gene and its expression product; (c) an ADGRF1 gene and its expression product; (d) CEACAM6 gene and its expression product; (e) CST6 gene and its expression product; (f) NDRG2 gene and its expression product; (g) MUC4 gene and its expression product; (h) PI3 gene and its expression product; (i) SDC1 gene and its expression product; (j) C15orf48 gene and its expression product.
2. The specific tumor cell molecular marker composition for promoting metastasis of ovarian cancer according to claim 1, wherein the specific tumor cell molecular marker composition is characterized by: the gene ID of NCBI database encoding S100A8 is 6279;
the gene ID of NCBI database coding S100A9 is 6280;
the gene ID of NCBI database encoding ADGRF1 is 266977;
the gene ID of NCBI database coding CEACAM6 is 4680;
the gene ID of NCBI database encoding CST6 is 1474;
the gene ID of the NCBI database coding NDRG2 is a sequence shown as 57447;
the gene ID encoding MUC4, NCBI database, is the sequence shown as 4585;
the gene ID of NCBI database encoding PI3 is 5266;
the gene ID of NCBI database encoding SDC1 is 6382;
the gene ID of NCBI database encoding C15orf48 is 84419;
the S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and C15orf48 genes and their expression products are all human sources.
3. Use of a reagent for identifying or detecting a specific tumor cell molecular marker composition for promoting metastasis of ovarian cancer according to claim 1 or 2 in the preparation of a medicament for diagnosing, detecting, monitoring or predicting the progression of ovarian cancer.
4. The use of claim 3, wherein the progression of ovarian cancer comprises proliferation and metastasis of ovarian cancer cells in vitro, and malignant growth in vivo.
5. A molecular composition kit for diagnosing, detecting, monitoring or predicting the progression of ovarian cancer, said kit comprising a reagent for identifying or detecting a specific tumor cell molecular marker composition for promoting metastasis of ovarian cancer according to claim 1 or 2.
6. The kit according to claim 5, wherein the reagent detects mRNA or cDNA of the specific tumor cell molecular marker composition for promoting metastasis of ovarian cancer according to claim 1 or 2 by a nucleic acid detection method; the nucleic acid detection method is transcription analysis, real-time fluorescence quantitative PCR, nucleic acid hybridization or any combination thereof; the reagents each independently comprise at least one primer or probe complementary to a portion of the nucleotide sequence of an mRNA or cDNA of the specific tumor cell molecular marker composition for promoting metastasis of ovarian cancer of the type described in claim 1 or 2.
7. The kit according to claim 6, wherein the kit comprises at least one or more of the following 10 primer pairs, and the primer pair sequences of the real-time quantitative reverse transcription PCR are shown in SEQ ID NO. 3-22.
8. A pharmaceutical composition for treating or preventing metastasis of ovarian cancer, comprising a substance that inhibits the S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and/or C15orf48 genes and expression products and/or reduced activity thereof, or any combination thereof.
9. The pharmaceutical composition of claim 8, wherein the agent that inhibits S100A8, S100A9, ADGRF1, CEACAM6, CST6, NDRG2, MUC4, PI3, SDC1 and/or C15orf48 genes and expression products and/or reduced activity thereof comprises a protein specific antibody, an RNA interfering molecule or antisense oligonucleotide directed against a gene mRNA, a small molecule inhibitor, siRNA or shRNA. The siRNA sequence is shown as SEQ ID NO.23-SEQ ID NO. 62.
10. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises an effective amount of a gene inhibitor and a pharmaceutically acceptable carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310912767.0A CN116769919A (en) | 2023-07-24 | 2023-07-24 | Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310912767.0A CN116769919A (en) | 2023-07-24 | 2023-07-24 | Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116769919A true CN116769919A (en) | 2023-09-19 |
Family
ID=88013452
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310912767.0A Pending CN116769919A (en) | 2023-07-24 | 2023-07-24 | Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116769919A (en) |
-
2023
- 2023-07-24 CN CN202310912767.0A patent/CN116769919A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109423517B (en) | Use of exosomes in tumor diagnosis, treatment and prognosis evaluation | |
| CN109468382B (en) | Application of lncRNA in diagnosis and treatment of lung adenocarcinoma | |
| Liu et al. | MicroRNA155 in the growth and invasion of salivary adenoid cystic carcinoma | |
| CN109593848A (en) | A kind of tumour correlated series, long-chain non-coding RNA and its application | |
| CN106834486A (en) | Osteosarcoma molecule diagnosis and treatment mark and its application | |
| CN109825587B (en) | Glioma prognostic marker CPVL and application thereof | |
| CN107312855B (en) | Gene related to laryngeal squamous cell carcinoma and application thereof | |
| Zhang et al. | Tumor-derived exosomal lincRNA ROR promotes angiogenesis in nasopharyngeal carcinoma | |
| CN111455056A (en) | lncRNA marker derived from adipose cell exosome and application and product thereof | |
| CN111621567A (en) | Marker for diagnosing liver cancer, detection reagent and application thereof | |
| CN115011697B (en) | Use of ALYREF in prostate cancer treatment and assessment | |
| CN105624325A (en) | Marker for diagnosing and treating lung adenocarcinoma | |
| CN116769919A (en) | Specific tumor cell molecular marker composition for promoting ovarian cancer metastasis and application thereof | |
| CN113293208B (en) | Molecular marker related to lung cancer proliferation and metastasis and application thereof | |
| CN111269987B (en) | Diagnostic prognosis marker MAPK8IP1P2 for thyroid cancer and application thereof | |
| CN112899371A (en) | Application of hsa _ circ _0000231 in treatment of tongue squamous cell carcinoma | |
| CN113908283A (en) | PRMT5 inhibitor and application thereof in combination with PD-L1 antibody blocking agent in treatment of lung cancer | |
| CN104403996B (en) | Human gastric cancer cell line with 5-fluorouracil resistance and establishment method and application thereof | |
| CN111088357B (en) | Tumor marker for ESCC and application thereof | |
| CN108130374B (en) | Diagnostic product and therapeutic pharmaceutical composition for kidney cancer | |
| CN106282385A (en) | Long-chain non-coding RNA XLOC_000090 qualification in pulmonary carcinoma and purposes | |
| CN107881240B (en) | The diagnosis and treatment marker of osteosarcoma | |
| CN112114143A (en) | Application of liver cancer diagnosis and cancer-causing kinase treatment marker | |
| CN110042164A (en) | Lung cancer diagnosis and treatment lncRNA marker | |
| CN105582536B (en) | Application of AGPAT9 gene in preparation of liver cancer treatment drug and diagnosis and prognosis evaluation reagent |
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 |