CN113679735A - Slc7a11基因在肝细胞癌介入栓塞术后中的应用 - Google Patents
Slc7a11基因在肝细胞癌介入栓塞术后中的应用 Download PDFInfo
- Publication number
- CN113679735A CN113679735A CN202110967726.2A CN202110967726A CN113679735A CN 113679735 A CN113679735 A CN 113679735A CN 202110967726 A CN202110967726 A CN 202110967726A CN 113679735 A CN113679735 A CN 113679735A
- Authority
- CN
- China
- Prior art keywords
- slc7a11
- mettl14
- hcc
- cells
- gene
- 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
- 206010073071 hepatocellular carcinoma Diseases 0.000 title claims abstract description 56
- 231100000844 hepatocellular carcinoma Toxicity 0.000 title claims abstract description 50
- 230000010102 embolization Effects 0.000 title claims abstract description 14
- 101150018817 SLC7A11 gene Proteins 0.000 title claims abstract description 8
- 102100035300 Cystine/glutamate transporter Human genes 0.000 claims abstract description 28
- 108091006241 SLC7A11 Proteins 0.000 claims abstract description 28
- 239000003112 inhibitor Substances 0.000 claims abstract description 13
- 108091027967 Small hairpin RNA Proteins 0.000 claims abstract description 11
- 239000004055 small Interfering RNA Substances 0.000 claims abstract description 11
- 230000035755 proliferation Effects 0.000 claims abstract description 10
- 206010027476 Metastases Diseases 0.000 claims abstract description 9
- 230000009401 metastasis Effects 0.000 claims abstract description 9
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 239000003814 drug Substances 0.000 claims description 4
- 108700011259 MicroRNAs Proteins 0.000 claims description 2
- 108091030071 RNAI Proteins 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000009368 gene silencing by RNA Effects 0.000 claims description 2
- 239000002679 microRNA Substances 0.000 claims description 2
- 102100031578 N6-adenosine-methyltransferase non-catalytic subunit Human genes 0.000 abstract description 81
- 101001013582 Homo sapiens N6-adenosine-methyltransferase non-catalytic subunit Proteins 0.000 abstract description 80
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 abstract description 34
- 230000000694 effects Effects 0.000 abstract description 29
- 206010021143 Hypoxia Diseases 0.000 abstract description 24
- 206010028980 Neoplasm Diseases 0.000 abstract description 23
- 229910052742 iron Inorganic materials 0.000 abstract description 17
- 230000034994 death Effects 0.000 abstract description 16
- 230000007954 hypoxia Effects 0.000 abstract description 15
- 230000002018 overexpression Effects 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 13
- 230000005764 inhibitory process Effects 0.000 abstract description 9
- 201000007270 liver cancer Diseases 0.000 abstract description 8
- 208000014018 liver neoplasm Diseases 0.000 abstract description 6
- 150000003384 small molecules Chemical class 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 3
- 238000013508 migration Methods 0.000 abstract description 3
- 230000005012 migration Effects 0.000 abstract description 3
- 230000006907 apoptotic process Effects 0.000 abstract description 2
- 238000013461 design Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 64
- 230000014509 gene expression Effects 0.000 description 32
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 29
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 23
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 23
- 108020004999 messenger RNA Proteins 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 15
- 238000003197 gene knockdown Methods 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 description 13
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 description 13
- 239000003642 reactive oxygen metabolite Substances 0.000 description 13
- 238000011529 RT qPCR Methods 0.000 description 12
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 11
- 101000725401 Homo sapiens Cytochrome c oxidase subunit 2 Proteins 0.000 description 10
- 101000605127 Homo sapiens Prostaglandin G/H synthase 2 Proteins 0.000 description 10
- 101000744745 Homo sapiens YTH domain-containing family protein 2 Proteins 0.000 description 10
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 10
- 102100039644 YTH domain-containing family protein 2 Human genes 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 108060001084 Luciferase Proteins 0.000 description 9
- 239000005089 Luciferase Substances 0.000 description 9
- 230000001146 hypoxic effect Effects 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 102000000905 Cadherin Human genes 0.000 description 8
- 108050007957 Cadherin Proteins 0.000 description 8
- 230000004614 tumor growth Effects 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 241000699660 Mus musculus Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 230000029663 wound healing Effects 0.000 description 5
- 108050000637 N-cadherin Proteins 0.000 description 4
- 102100040619 N6-adenosine-methyltransferase catalytic subunit Human genes 0.000 description 4
- 108010065472 Vimentin Proteins 0.000 description 4
- 102000013127 Vimentin Human genes 0.000 description 4
- 230000035508 accumulation Effects 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 238000001493 electron microscopy Methods 0.000 description 4
- 230000003859 lipid peroxidation Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 210000005048 vimentin Anatomy 0.000 description 4
- 108020003589 5' Untranslated Regions Proteins 0.000 description 3
- 108010092160 Dactinomycin Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 101000967135 Homo sapiens N6-adenosine-methyltransferase catalytic subunit Proteins 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 229960000640 dactinomycin Drugs 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 230000005760 tumorsuppression Effects 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- VQAYFKKCNSOZKM-IOSLPCCCSA-N N(6)-methyladenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VQAYFKKCNSOZKM-IOSLPCCCSA-N 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 2
- 102100023345 Tyrosine-protein kinase ITK/TSK Human genes 0.000 description 2
- 102100023905 YTH domain-containing protein 1 Human genes 0.000 description 2
- 229960001570 ademetionine Drugs 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine group Chemical group [C@@H]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(N)=NC=NC12 OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000002991 immunohistochemical analysis Methods 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 238000003468 luciferase reporter gene assay Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000001617 migratory effect Effects 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 102100033400 4F2 cell-surface antigen heavy chain Human genes 0.000 description 1
- 101150000157 ARHGEF1 gene Proteins 0.000 description 1
- 206010006002 Bone pain Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 101100297347 Caenorhabditis elegans pgl-3 gene Proteins 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 1
- 238000003718 Dual-Luciferase Reporter Assay System Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 240000004153 Hibiscus sabdariffa Species 0.000 description 1
- 235000001018 Hibiscus sabdariffa Nutrition 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001012535 Homo sapiens N(6)-adenine-specific methyltransferase METTL4 Proteins 0.000 description 1
- 101000914035 Homo sapiens Pre-mRNA-splicing regulator WTAP Proteins 0.000 description 1
- 101000666873 Homo sapiens Protein virilizer homolog Proteins 0.000 description 1
- 101000976373 Homo sapiens YTH domain-containing protein 1 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 101100467856 Mus musculus Rbmy1a1 gene Proteins 0.000 description 1
- 101100467858 Mus musculus Rbmy1b gene Proteins 0.000 description 1
- 102100029738 N(6)-adenine-specific methyltransferase METTL4 Human genes 0.000 description 1
- 101710158306 N6-adenosine-methyltransferase catalytic subunit Proteins 0.000 description 1
- 101710081491 N6-adenosine-methyltransferase non-catalytic subunit Proteins 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102100026431 Pre-mRNA-splicing regulator WTAP Human genes 0.000 description 1
- 102100038288 Protein virilizer homolog Human genes 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108091006313 SLC3A2 Proteins 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 229910052770 Uranium Inorganic materials 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 101710084664 YTH domain-containing protein 1 Proteins 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008995 epigenetic change Effects 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000000506 liquid--solid chromatography Methods 0.000 description 1
- 101150055452 lsc gene Proteins 0.000 description 1
- 230000017156 mRNA modification Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000013152 negative regulation of cell migration Effects 0.000 description 1
- 230000006610 nonapoptotic cell death Effects 0.000 description 1
- 230000007959 normoxia Effects 0.000 description 1
- 201000002740 oral squamous cell carcinoma Diseases 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000003571 reporter gene assay Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000002473 ribonucleic acid immunoprecipitation Methods 0.000 description 1
- 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 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 238000013414 tumor xenograft model Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了SLC7A11基因在肝细胞癌介入栓塞术后中的应用。本发明基于分子生物学实验验证了SLC7A11为铁死亡信号通路的关键成员,并针对SLC7A11及相关信号分子通路,发现和设计相关的小分子抑制剂,干预肝癌细胞增殖凋亡;本发明通过动物模型实验验证了小分子抑制剂(SLC7A11的shRNA)敲低SLC7A11能够抑制HCC细胞的增殖和迁移,而SLC7A11的过表达可以显著挽救METTL14在HCC缺氧条件下诱导的肿瘤抑制作用,为临床上肝癌介入栓塞术后抑制HCC的复发转移提供了新的分子靶标。
Description
技术领域
本发明涉及SLC7A11基因在肝细胞癌介入栓塞术后中的应用,属于分子生物技术领域。
背景技术
肝细胞癌(HCC)是最常见的原发性肝癌,其典型特征是快速增殖和转移较早。巴塞罗那临床肝癌(BCLC)标准和中国原发性肝癌诊治指南(2017版)都推荐了不同阶段HCC的治疗方法。然而,临床上仍需要更多治疗方法,因为该疾病通常在中晚期被诊断出来,并且HCC患者的五年生存率仍不理想。
介入治疗已广泛应用于不可切除的HCC患者。然而,介入栓塞引起的缺氧状态可能促进HCC细胞的增殖和转移,其内在机制尚不清楚。因此,探索缺氧与HCC发展的分子机制对于推进未来的治疗至关重要。值得注意的是,异常的表观遗传变化会导致基因表达的严重破坏,从而促进HCC的发生和发展。
N6-甲基腺苷(m6A)RNA修饰已成为表观遗传调控机制的一个新维度,可在翻译前控制mRNA表达。在至少三分之一的哺乳动物mRNA中发现了 m6A。据估计,一个mRNA中平均有3-5个m6A修饰。值得注意的是,许多 m6A位点在小鼠和人类之间在进化上是保守的。多组分m6A甲基转移酶复合物(MTC)在mRNA修饰中执行m6A的沉积,其由异二聚体、甲基转移酶样3(METTL3)/甲基转移酶样14(METTL14)复合物、主要酶复合物和其他参与因素包括KIAA1429、WTAP和RBM15。就该复合体而言,METTL3是与甲基供体S-腺苷甲硫氨酸(SAM)结合并催化甲基基团转移的催化亚基,而METTL14 通过稳定METTL3构象和识别底物RNA来负责m6A沉积。m6A修饰位点经常在3'非翻译区(3'UTR)和编码序列(CDS)中富集,在终止密码子区域周围富集特别高,其中包含经典的共有序列DRACH(D=G、A或U;R=G或A; H=A、C或U).这种可逆的催化过程由m6A橡皮擦脂肪量和肥胖相关蛋白 (FTO)和alkB同源物5(ALKBH5)进行。m6A修饰的RNA可以通过m6A 阅读器蛋白进行识别,包括YT521-B同源(YTH)域家族蛋白 (YTHDF1–YTHDF3、YTHDC1和YTHDC2)。其中,YTHDF2是第一个经过鉴定和充分研究的m6A阅读器蛋白,它通过其C端YTD结构域靶向 m6A,N端结构域负责将mRNA推向加工体以进一步降解。
最近的研究表明m6A参与了多种生理过程。作为MTC的另一个不可或缺的组成部分,METTL14已被证明在各种类型的癌症中上调。Weng等人报道,敲除METTL14可以显着抑制白血病干/起始细胞(LSCs/LICs)的自我更新。进一步的机制研究表明,其靶标mRNAMYB和MYC上m6A丰度的降低导致 mRNA稳定性和翻译的降低。据报道,METTL14还可以驱动EBV介导的肿瘤发生。然而,Chen等人发现METTL14通过靶向miR-37528抑制结直肠癌 (CRC)进展;Ma等人认为METTL14通过靶向pri-miR-12629抑制HCC转移。然而,METTL14在肝癌介入栓塞术后的肿瘤复发转移中的具体功能仍然难以捉摸。
铁死亡是以一种以脂质过氧化产物(MDA)和/或致死活性氧(ROS)增多、铁依赖性为主要特征的非凋亡性细胞死亡方式,在癌症治疗中显示出巨大的前景。由SLC7A11和SLC3A2组成的xc-系统的功能是输入胞外氧化形式的半胱氨酸、胱氨酸,输出胞内谷氨酸以保持氧化还原平衡。在一些癌症患者中检测到更高水平的SLC7A11,抑制SLC7A11会使肿瘤细胞对铁死亡敏感。Ungard等人发现乳腺癌细胞中SLC7A11的沉默延迟了癌症诱导的骨痛发作;Li等人预测SLC7A11的过表达与口腔鳞状细胞癌患者的复发呈正相关。所有这些结果表明xc-系统抑制剂可能作为潜在应用前景的抗癌剂。然而,我们对HCC中铁死亡调节的确切机制的了解仍不清楚,尤其是在缺氧条件下。m6A机制的破坏是否会导致铁死亡细胞死亡并促进HCC发病机制仍需要探索。
发明内容
本发明所要解决的技术问题是:肝细胞癌介入栓塞术后由引起的缺氧状态可能促进HCC细胞的增殖和转移等问题。
为了解决上述技术问题,本发明公开了SLC7A11基因在肝细胞癌介入栓塞术后中的应用,包括将SLC7A11抑制剂用于制备肝细胞癌介入栓塞术后抑制 HCC细胞的增殖和转移的药物中的应用。
优选地,所述药物包括医学上可接受的载体和有效量的活性成分,所述活性成分为SLC7A11抑制剂。
优选地,所述的SLC7A11抑制剂包括SLC7A11基因特异性的RNAi、 SLC7A11基因特异性的microRNA、SLC7A11基因的shRNA或SLC7A11基因的siRNA。
优选地,所述的SLC7A11抑制剂为SLC7A11基因的shRNA,所述shRNA 的序列为TTCTCCGAACGTGTCA CGTTTC。
与现有技术相比,本发明的有益效果在于:
1.本发明基于分子生物学实验验证了SLC7A11为铁死亡信号通路的关键成员,并针对SLC7A11及相关信号分子通路,发现和设计相关的小分子抑制剂,干预肝癌细胞增殖凋亡;
2.本发明通过动物模型实验验证了小分子抑制剂(SLC7A11的shRNA)敲低SLC7A11能够抑制HCC细胞的增殖和迁移,而SLC7A11的过表达可以显著挽救METTL14在HCC缺氧条件下诱导的肿瘤抑制作用,为临床上肝癌介入栓塞术后抑制HCC的复发转移提供了新的分子靶标。
附图说明
图1A:表示缺氧下调Huh7和HCCLM3细胞中的METTL14;
图1B:表示HIF-1α敲低对缺氧(Hypoxia)诱导的METTL14抑制的影响;
图1C:表示通过流式细胞术检测到的HIF-1α敲低对缺氧诱导的ROS积累的影响以及量化结果;
图1D:表示HIF-1α敲低对缺氧诱导的MDA积累的影响;
图1E:表示电子显微镜检测线粒体形态变化,其中,白色箭头指的是典型的线粒体;
图2A:表示Oncomine(https://www.oncomine.org/resource/main.html)分析的“Guichard Liver”和“Guichard Liver 2”数据库和OncoLnc(http://www.oncolnc.org)分析的TCGA数据库的生物信息学分析/)关键字为“METTL14”,“肝细胞癌与正常分析”;
图2B:表示在Oncomine(https://www.oncomine.org/resource/main.html)使用关键词“SLC7A11”、“肝细胞癌”分析了来自“Wurmbach Liver”、“Roessler Liver”和“Roessler Liver 2”数据库的生物信息学分析对比正态分析”;
图2C:表示METTL14在七种HCC细胞系(Huh7、HepG2、7721、HCCLM3、 MHCC97H、PLC/PRF/5、Bel-7402)中的蛋白表达模式,与通过蛋白质印迹检测的正常肝细胞系L02进行比较(下图),相对METTL14蛋白质水平被量化(上图);
图2D:表示与正常肝细胞系L02进行比较,分别通过qPCR和蛋白质印迹检测的七种HCC细胞系(Huh7、HepG2、SMMC-7721、HCCLM3、MHCC97H、 PLC/PRF/5、Bel-7402)中SLC7A11的mRNA和蛋白水平;
图2E:表示METTL14对缺氧条件下Huh7和HCCLM3细胞SLC7A11表达的影响,分别通过RT-qPCR和蛋白质印迹检测SLC7A11 mRNA和蛋白质水平;
图3A:表示SLC7A11 mRNA的示意图和5'UTR预测的“m6A”位点(图中圈出的碱基A),“DRACH”中间的碱基A被T替换,以制备用于荧光素酶报告基因检测的突变质粒;
图3B:表示METTL14和METTL14-R298P突变体对HCCLM3细胞中SLC7A11表达的影响,分别通过qPCR和Western印迹检测SLC7A11的mRNA 和蛋白质水平;
图3C:表示DOT BLOT显示稳定表达宽型METTL14和 METTL14-R298P突变体的总m6A水平;
图3D:表示SLC7A11荧光素酶报告基因示意图;
图3E:表示确定了基于pGL3基本质粒的WT或MUT荧光素酶报告基因在METTL14转染的HCCLM3细胞中的相对活性(标准化为载体对照组);
图3F:表示应用MeRIP分析和qRT-PCR来评估HCCLM3表达的宽型 METTL14或METTL14-R298P突变体中SLC7A11的m6A修饰,通过 m6A-IP/input和IgG-IP/input计算各组m6A的富集度;
图3G:表示宽型METTL14和METTL14-R298P突变体对HCC肿瘤生长的影响,裸鼠皮下注射稳定表达METTL14、METTL14-R298P或对照载体的 HCCLM3细胞,每周计算两次肿瘤生长;
图3H:表示在异种移植模型中过表达HCCLM3细胞(或阴性对照)的稳定宽型METTL14或METTL14-R298P突变体的肿瘤生长曲线;
图3I:表示通过蛋白质印迹检测异种移植物中COX2、SLC7A11和 METTL14的表达模式;
图3J:表示种移植物中METTL14分别和COX2、SLC7A11之间的相关性;
图3K:表示三种异种移植物的H&E染色切片;
图3L:表示免疫组织化学检测的异种移植物中COX2、SLC7A11和 METTL14的表达模式;
图4A:表示在用放线菌素D(标准化为0小时)处理后,在Huh7和 HCCLM3细胞中测定了mRNA衰减率;
图4B:表示RT-qPCR和蛋白质印迹显示沉默YTHDF2对Huh7和 HCCLM3中SLC7A11mRNA和蛋白质水平的影响;
图4C:表示转染了siYTHDF2的Huh7和HCCLM3细胞中WT或 MUT荧光素酶报告基因的相对活性(标准化为载体对照组)的测定结果;
图5A:表示根据制造商的说明通过流式细胞术进行ROS检测的定量分析结果;其中,Huh7和HCCLM3细胞用或不用10mM浓度的NAC处理36小时;
图5B:表示通过蛋白质印迹和qPCR分别检测Huh7和HCCLM3细胞中EMT 相关E-cadherin、N-cadherin和Vimentin的蛋白和mRNA水平,其中,细胞用或不用10mM浓度的NAC处理36小时;
图5C:表示通过电子显微镜检测HCCLM3细胞中用/不用NAC或 shSLC7A11处理的线粒体的形态学改变,其中,白色箭头指的是典型的线粒体;
图5D:表示SLC7A11敲低对肿瘤生长的影响以及在5周的时间过程中检测的肿瘤体积生长曲线;
图5E:表示肿瘤切片的H&E染色图,显示为肿瘤结构;
图5F:表示免疫组织化学分析显示shSLC7A11组具有更高的COX2表达;
图6A:表示蛋白质印迹显示了Huh7和HCCLM3细胞中METTL14和 SLC7A11的表达模式,其中,稳定表达宽型METTL14或R298P突变体的细胞用SLC7A11过表达(转染);
图6B:表示伤口愈合显示METTL14对照、METTL14过表达和 METTL14-R298P以及SLC7A11分别对缺氧条件下Huh7和HCCLM3细胞的迁移率的影响;
图6C:表示CCK-8测定说明了METTL14对照、METTL14过表达和 METTL14-R298P,加上SLC7A11在缺氧条件下Huh7和HCCLM3细胞的增殖趋势;
以上各图中,“NS”:表示不具有显著性差异(not significant);*:表示p<0.05;**:表示p<0.01;***:表示p<0.001;****:表示p<0.0001。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
以下实施例中,所用到的具体实验方法如下所示:
(1)细胞培养及处理
七株人肝癌细胞系(Huh7、HepG2、7721、HCCLM3、MHCC97H、PLC/PRF/5 和Bel-7402)和一株正常人肝细胞株L02来自中山医院张士哲博士和甘伟博士,复旦大学(中国上海)。所有细胞系均在Dulbecco改良的Eagle培养基(DMEM,Gibco,Grand Island,NY,USA)中培养。DMEM的额外混合物是10%胎牛血清(FBS)、抗生素(青霉素(100U/ml)/链霉素(0.1mg/ml))。培养细胞需要5%CO2、37℃的湿润环境。培养基每周更新两次。N-乙酰-L-半胱氨酸(NAC)购自Apexbio(美国休斯顿)。
(2)转染和稳定细胞株
Lipofectamine 2000试剂(Life Technology,Thermo Fisher Scientific,DE,USA)用于进行瞬时转染。pcDNA3.1-METTL14、pcDNA3.1-YTHDF2、 pLKO.1-SLC7A11和pcDNA3.1-SLC7A11-His(+)购自Genechem(中国上海)。空载体用作阴性对照。根据制造商的说明使用Lipofectamine 2000试剂 (Invitrogen)进行转染测定。本实验共使用5×105个细胞。转染48小时后,通过RT-qPCR分析或蛋白质印迹验证转染效率。
(3)实时荧光定量PCR
根据制造商的说明,通过RNA纯化试剂盒(EZbioscience,USA)提取总 RNA。然后,使用4×Reverse Transcription Master Mix(EZbioscience)进行RT-PCR,使用无DNAase和RNAase的尖端(YueYiBioTech,上海,中国)。 SYBR Green PCR试剂盒(Yeasen,中国)用于qPCR。根据2-ΔΔCt方法,将每个基因的表达水平标准化为GAPDH的表达水平,作为内部对照。METTL14、YTHDF2、SLC7A11、E-钙粘蛋白、N-钙粘蛋白、波形蛋白和GAPDH 的引物(Sunya,中国)如下:
METTL14(F:5′-CATCAGGCTAAAGGATGAGTT-3′(SEQ ID NO:1);R:5′-CTAACTTCATAATATCATCC-3′(SEQ ID NO:2));
YTHDF2(F:5′-AGCCCCACTTCCTACCAGATG-3′(SEQ ID NO:3);R:5′-TGAGAACTGTTATTTCCCCATGC-3′(SEQ ID NO:4));
SLC7A11(F:5′-GTCTGGAGAAACAGCCAAGG-3′(SEQ ID NO:5);R: 5′-CGGAGTTCCTCGAATAGCTG-3′(SEQ ID NO:6));
E-cadherin(F:5′-CGAGAGCTACACGTTCACGG-3′(SEQ ID NO:7);R:5′-GGGTGTCGAGGGAAAAATAGG-3′(SEQ ID NO:8));
N-cadherin(F:5′-CTGACAATGACCCCACAGC-3′(SEQ ID NO:9);R: 5′-TCCTGCTCACCACCACT ACTT-3′(SEQ ID NO:10));
Vimentin(F:5′-TCTACGAGGAGGAGATGCGG-3′;(SEQ ID NO:11)R: 5′-GGTCAAGACGTGCCAG AGAC-3′(SEQ ID NO:12));
GAPDH(F:5′-GCACCGTCAAGGCTGAGAAC-3′(SEQ ID NO:13);R: 5′-TGGTGAAGACGCCAGTGGA-3′(SEQ ID NO:14))。
(4)细胞增殖实验
根据制造商的方案,通过Cell Counting Kit-8测定(Yeason,Shanghai,China)检测细胞增殖。根据制造商的方案,在每个孔中种植10000个细胞24小时或 12小时。检查点结束时,用10μl CCK8溶液检测每孔的OD值。在450nm和 600nm处用分光光度法测量溶液。
(5)蛋白免疫印迹
RIPA收集细胞,每组取总蛋白20μg进行电泳。将膜与5%脱脂牛奶在 22℃下孵育2小时,然后与一抗孵育。主要抗体如下:Actin(13E5,CST), METTL14(D8K8W,CST),YTHDF2(ab220163,abcam),SLC7A11(ab175186, abcam),E-cadherin(24E10,CST),N-cadherin(D4R1H)CST)、波形蛋白(D21H3, CST)和肌动蛋白(13E5,CST)在4℃下过夜。随后,在22℃温度下使用抗兔二抗2小时。使用化学发光ECL试剂盒(Tanon,上海,中国)观察蛋白质条带。
(6)划痕实验
为了评估细胞的迁移能力,进行了伤口愈合试验。如果需要,将NAC和/ 或pcDNA3.1-SLC7A11-His(+)(2μg)添加到孔中。细胞在37℃和5%CO2条件下培养。用20-200μl移液器吸头进行划痕,随后用PBS洗涤板并更换为新鲜培养基。使用相差显微镜(佳能,日本)在划伤后0和48小时拍摄伤口愈合图像。
(7)活性氧ROS检测
为了评估细胞的迁移能力,进行了伤口愈合试验。如果需要,将NAC和/ 或pcDNA3.1-SLC7A11-His(+)(2μg)添加到孔中。细胞在37℃和5%CO2条件下培养。用20-200μl移液器吸头进行划痕,随后用PBS洗涤板并更换为新鲜培养基。使用相差显微镜(佳能,日本)在划伤后0和48小时拍摄伤口愈合图像。
(8)荧光报告基因实验
根据制造商的说明(11402ES60,Yeasen)收集RNA。Huh7或HCCLM3 细胞接种在6孔板中,并用宽型SLC7A11响应荧光素酶报告构建体 (SLC7A11-WT)、突变体SLC7A11响应荧光素酶报告构建体(SLC7A11-MUT)、宽型或METTL14质粒转染,siYTHDF2,相应地。在转染后24h,将细胞裂解物与10μg/ml的萤火虫和TK分别孵育10分钟。通过双荧光素酶报告基因检测系统(Promega,Madison,WI,USA)和微孔板光度计(Promega)测量荧光素酶活性。萤火虫荧光素酶活性被相应的海肾荧光素酶活性校正。
(9)甲基化RNA免疫沉淀(MeRIP)
MeRIP遵循制造商的说明(PierceTM Magnetic RNA-Protein Pull-Down Kit,Cat.No.20164)。简而言之,分离出200μg的总RNA用于polyA+RNA (Promega)并进行定量。PolyA+RNA被分成大约100nt长的片段。在进行 m6A-IP之前,使用生物分析仪确保RNA片段化。之后,首先进行第一链cDNA 合成。qPCR如所述进行。
(10)RNA稳定性实验
如前所述进行RNA衰变测定。简而言之,将Huh7和HCCLM3细胞以 60%的汇合度接种在6厘米的平板中。24小时后,将每个6厘米的平板重新播种到三个6厘米的平板中。48小时后,在收集前的8小时、4小时、2小时和0小时加入放线菌素D至3mg/ml。通过柱上DNase-I消化步骤纯化总 RNA。RNA量由RT-qPCR确定。
(11)MDA实验
为了测试脂质过氧化,我们使用脂质过氧化(MDA)检测试剂盒(货号 MAK085,Sigma-Aldrich,美国)。该程序遵循制造商的说明。
(12)电镜拍照
为了获得线粒体在细胞中的位置,对不同干预的HCC细胞进行电子显微镜摄影。为了成像,如下制备细胞。样品在4℃下用1%锇酸固定1小时。之后,使用ddH2O清洗样品。然后,样品用醋酸铀染色过夜。使用不同浓度的酒精使样品脱水。样品经EMBED 812EMBEDDINGKIT包埋,最后聚合成像。
(13)动物模型
使用不同的慢病毒感染野生型HCCLM3细胞株,具体使用的慢病毒种类有:SLC7A11-Control(无义序列)、SLC7A11-Knockdown(shRNA的序列为: TTCTCCGAACGTGTCACGTTTC)(SEQ ID NO:15);METTL14-Vector(无义序列)、METTL14-Overexpression、METTL14-R298P共五种慢病毒。根据说明书,准备好5×105个HCCLM3细胞,使用额定MOI(滴度单位)的对应病毒感染细胞,并于37℃,5%CO2,90%以上湿度的条件下培养。18小时后,使用嘌呤霉素进行稳转株的筛选(载体均带有表达GFP及嘌呤抗性的片段)。3天后在荧光显微镜下使用约488nm处的单个激发峰,发射峰波长为509nm的荧光观察稳转株的建立。确认几乎所有细胞均带有绿色荧光后,再次予以细胞扩增。
成功建立稳转株后,进行皮下瘤种植。本研究主要分为两个动物实验,分别有二组和三组不同类别的皮下瘤移植瘤模型。(1):METTL14-Vector、 METTL14-Overexpression、METTL14-R298P三种移植瘤观察METTL14的m6A 功能对肿瘤生长的作用;(2)SLC7A11-Control、SLC7A11-Knockdown两种移植瘤观察SLC7A11对肿瘤生长的作用。
具体种植过程为:消化每种细胞,以3.5×107个细胞溶解于100μl的PBS 中,并注射于裸鼠背侧靠右后皮下;7天后观察成瘤情况并进行下一步实验;约 5周后处死裸鼠,最终根据定期(每3天)测量结果对移植瘤进行计算并绘制肿瘤生长图。
(14)H&E染色和免疫组织化学
异种移植样品在4%多聚甲醛(Sigma-Aldrich,DK-2860,丹麦)中固定过夜,并在切割6μm切片之前嵌入石蜡中。抗原修复在100℃的柠檬酸盐缓冲液中进行25分钟。将0.1%Tween20和2.5%BSA(Sigma-Aldrich)混合到样品中。一抗如下:METTL14(ab220030,Abcam,U.K.)、SLC7A11(ab37185,Abcam, U.K.)和COX2(ab179800,Abcam,U.K.)。应用适当的辣根过氧化物酶偶联二抗进行开发(ab205718,Abcam,英国)。
实施例1
缺氧在体外以HIF-1α依赖性方式抑制METTL14诱导的铁死亡:
为了确定缺氧条件下METTL14和RNA m6A修饰的作用,首先检测了 METTL14在含1%O2(缺氧条件,Hypoxia)的Huh7和HCCLM3细胞系中的表达。结果表明,与对照组(常氧,Normoxia)相比,缺氧有效地降低了 METTL14并增加了两种HCC细胞系中的HIF-1α表达,如图1A所示。然后,采用shRNA敲低HIF-1α,结果表明对HIF-1α的抑制强烈阻止了缺氧诱导的METTL14下调,如图1B所示,这表明缺氧引发的METTL14抑制是HIF-1α依赖性的。铁死亡是一种受调节的细胞死亡形式,已被证明具有肿瘤抑制功能,可用于癌症治疗。ROS和MDA是铁死亡的公认指标。为了探讨缺氧对铁死亡的影响,通过流式细胞术测定ROS(活性氧)。结果显示抑制HIF-1α增强了 Huh7和HCCLM3细胞系中ROS的积累(p<0.0001),如图1C所示。此外,MDA(脂质过氧化)测定显示,与对照组相比,HIF-1α敲低组的MDA含量更高(p<0.01),如图1D所示。此外,众所周知,铁死亡可以诱导典型的形态变化,其特征是线粒体浓缩和破坏。一致地,与对照组相比,在实验中观察到敲低 HIF-1α显著诱导更小、更密集的线粒体膜受损,如图1E所示。以上实验结果表明缺氧以HIF-1α依赖性方式下调METTL14,并通过敲低HIF-1α可诱导 HCC细胞中的铁死亡。
实施例2
METTL14在HCC中负调节SLC7A11的表达:
为了探索METTL14调节铁死亡的潜在机制,使用多个数据库分析了 METTL14和SLC7A11的表达模式,该系统是介导铁死亡的系统xc-的核心成员。首先,在癌症基因组图谱(TCGA)和Oncomine数据库中分析了METTL14 在HCC患者与健康患者中的表达模式。与正常肝脏相比,METTL14在HCC 患者中的表达水平分别下调1.052倍(p=7.32×10-9)、1.059倍(p=4.86×10-4);另一方面,METTL14表达较低的HCC患者的总生存期短于METTL14表达较高的患者,如图2A所示。此外,在Oncomine数据库“Wurmbach Liver”、“Roessler Liver”和“Roessler Liver 2”中进一步分析了SLC7A11在HCC患者和健康受试者中的表达。与健康受试者相比,HCC患者的SLC7A11的表达上调至 3.343倍(p=6.18×10-6)、1.943倍(p=1.58×10-4)、1.494倍(p=1.47×10-18),如图2B所示。
在7种HCC细胞系中进一步分析了METTL14表达。与正常肝细胞系 L02相比,除HepG2外,Huh7、7721、HCCLM3、MHCC97H、PLC/PRF/5和 Bel-7402细胞系中METTL14蛋白降低,如图2C所示。
另外,除了HepG2和MHCC97H外,大多数检测到的HCC细胞系中SLC7A11的mRNA和蛋白质水平均显著上调,如图2D所示,表明METTL4 可能与SLC7A11表达呈负相关。重要的是,缺氧条件下过表达的METTL14可以在mRNA和蛋白质水平上显著下调SLC7A11的表达,如图2E所示。以上结果表明,METTL14负调节SLC7A11表达。
实施例3
METTL14在HCC中SLC7A11 mRNA的5'UTR处触发m6A甲基化:
为了验证SLC7A11和METTL14之间的具体关系,首先检查了RNA Base v2.0(http://www.sysu.edu.cn),发现SLC7A11 mRNA的5'UTR内有几个潜在的 m6A位点,如图3A所示,这表明SLC7A11可能以依赖于m6A的方式由 METTL14调节。已经证明R298P突变大大降低了METTL14甲基化活性。因此建立了稳定的METTL14-R298P表达Huh7和HCCLM3细胞系,如图3B 所示。为了确定SLC7A11的m6A修饰是否由METTL14介导,首先通过 m6A斑点印迹法检测了阴性对照组和稳定的METTL14过表达组以及 METTL14-R298P组中的总m6A水平。正如预期的那样,m6A水平随着 METTL14的过表达而显著增加,但在两个HCC细胞系中因R298P突变而降低,如图3C所示。
为了探索SLC7A11上m6A修饰的本质,使用野生型(WT)和突变型 (MUT)质粒进行荧光素酶报告基因检测,如图3D所示。对于突变报告基因,胞嘧啶碱基(C)被设计为在几个预测的m6A位点替换腺苷碱基(A)以阻断 m6A甲基化的影响,而野生型报告基因包含完整的m6A位点。正如预期所见, METTL14过表达适度降低了宽型组的荧光素酶活性,但对突变对应物几乎没有影响,如图3E所示,表明SLC7A11调节受METTL14引导的m6A修饰的控制。
此外,通过MeRIP-qPCR测定检测到SLC7A11中m6A的富集。与IgG 对照组相比,在m6A特异性抗体处理组中检测到SLC7A11转录物的显著富集。此外,METTL14-R298P突变表达后由m6A修饰的SLC7A11水平显著降低,如图3F所示。因此,METTL14可能会影响m6A的整体水平,特别是 SLC7A11。
为了证实METTL14在体内的作用,通过将具有宽型METTL14或 METTL14-R298P突变体稳定过表达的HCC细胞(HCCLM3)皮下注射到裸鼠中来构建肿瘤异种移植模型。结果发现宽型METTL14过表达抑制了肿瘤发生,与对照组相比,肿瘤体积显著降低。同时,METTL14-R298P突变体的强制表达在异种移植小鼠中失去了肿瘤抑制作用,如图3G,3H所示。一致地,与对照和METTL14-R298P突变组相比,METTL14过表达组表现出较低水平的 SLC7A11和较高水平的COX2,这是异种移植物中铁死亡的金指标,如图3I 所示。并且还显示了异种移植物中METTL14和SLC7A11之间呈负相关, METTL14和COX2之间呈正相关,如图3J所示。此外,肿瘤切片的H&E染色表示为肿瘤结构,如图3K所示。通过免疫组织化学进一步研究了异种移植肿瘤切片中METTL14、SLC7A11和COX2的表达。与其他两组相比,METTL14 过表达组表现出较低的SLC7A11表达和较高的COX2表达,如图3L所示。
因此,以上结果表明METTL14通过在HCC中以m6A依赖性方式靶向 SLC7A11来发挥肿瘤抑制功能。
实施例4
METTL14诱导的SLC7A11 mRNA衰减依赖于m6A-YTHDF2:
找出SLC7A11的阅读器至关重要,因为m6A修饰的mRNA转录物依赖于阅读器蛋白在功能上参与生物过程。在实施例2中的结果表明METTL14过表达显著下调了Huh7和HCCLM3细胞系中的SLC7A11 mRNA(图2E)。接下来测试了m6A修饰是否影响SLC7A11的mRNA稳定性。qPCR表明,在放线菌素D存在下,METTL14的过表达显著增强了SLC7A11 mRNA的降解,如图4A所示。
YTHDF2(YTH域家族2)是一种公认的m6A阅读器蛋白,已被证明可以调节mRNA的稳定性。通过敲低YTHDF2的表达在mRNA和蛋白质水平上显著增加了SLC7A11的表达,如图4B所示,这表明YTHDF2在SLC7A11 调节中的潜在作用。此外,如前所述,荧光素酶报告基因检测是用含有野生型 (WT)或突变体(MUT)SLC7A11的质粒进行的。正如预期的那样,YTHDF2的敲低显著增加了宽型组的荧光素酶活性,但对突变组几乎没有影响,如图4C所示。
因此,以上实验结果表明m6A-YTHDF2进行METTL14诱导的 SLC7A11 mRNA降解。
实施例5
SLC7A11的敲低刺激铁死亡并在HCC中表现出抗肿瘤作用:
为了验证SLC7A11抑制是否可以模拟METTL14的肿瘤抑制功能,首先构建了慢病毒敲低SLC7A11的HCCLM3细胞(shRNA的序列为: TTCTCCGAACGTGTCA CGTTTC(SEQ ID NO:15)),并检测了SLC7A11敲低对铁死亡诱导的影响,如图5A所示,shSLC7A11强烈刺激了ROS的产生,而ROS清除剂N-乙酰-L-半胱氨酸(NAC)在Huh7和Huh7中显著阻断了 HCCLM3细胞中shSLC7A11诱导的ROS积累以及EMT逆转,如图5B所示。此外,NAC治疗有效地消除了电子显微镜检测到的shSLC7A11诱导的线粒体收缩。与shSLC7A11组相比,shSLC7A11+NAC组的线粒体表现出相对更完整的膜和更大的尺寸,如图5C所示。此外,在裸鼠体内观察到同样的趋势。与对照组相比,SLC7A11的下调显著抑制了肿瘤生长,如图5D所示。肿瘤切片的H&E染色表示为肿瘤结构,如图5E所示。此外,免疫组织化学分析显示shSLC7A11组具有更高的COX2表达,如图5F所示。因此,通过抑制HCC细胞中的SLC7A11验证了抗肿瘤作用。
实施例6
外源性表达的SLC7A11消除了METTL14在HCC缺氧条件下诱导的肿瘤抑制作用:
通过进一步的实验检查了SLC7A11的抑制是否有助于METTL14的抗肿瘤作用。如图6A所示,在缺氧环境下,宽型METTL14而不是METTL14-R298P 突变体强烈抑制SLC7A11表达,而SLC7A11的过表达有效地消除了 METTL14在两种HCC细胞系中诱导的SLC7A11下调。正如预期的那样,由此产生的SLC7A11过表达明显消除了METTL14诱导的细胞迁移抑制,如图6B所示。此外,SLC7A11过表达还显著阻止了METTL14在Huh7和HCCLM3细胞中诱导的生长抑制,如图6C所示。以上实验结果表明SLC7A11 有效地参与了METTL14调节的生长和迁移。
上述实施例仅为本发明的优选实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
序列表
<110> 复旦大学附属中山医院
<120> SLC7A11基因在肝细胞癌介入栓塞术后中的应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
catcaggcta aaggatgagt t 21
<210> 2
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctaacttcat aatatcatcc 20
<210> 3
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
agccccactt cctaccagat g 21
<210> 4
<211> 23
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgagaactgt tatttcccca tgc 23
<210> 5
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtctggagaa acagccaagg 20
<210> 6
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
cggagttcct cgaatagctg 20
<210> 7
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 7
cgagagctac acgttcacgg 20
<210> 8
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 8
gggtgtcgag ggaaaaatag g 21
<210> 9
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctgacaatga ccccacagc 19
<210> 10
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcctgctcac caccactact t 21
<210> 11
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 11
tctacgagga ggagatgcgg 20
<210> 12
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggtcaagacg tgccagagac 20
<210> 13
<211> 20
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 13
gcaccgtcaa ggctgagaac 20
<210> 14
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 14
tggtgaagac gccagtgga 19
<210> 15
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 15
ttctccgaac gtgtcacgtt tc 22
Claims (4)
1.SLC7A11基因在肝细胞癌介入栓塞术后中的应用,其特征在于,包括将SLC7A11抑制剂用于制备肝细胞癌介入栓塞术后抑制HCC细胞的增殖和转移的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述药物包括医学上可接受的载体和有效量的活性成分,所述活性成分为SLC7A11抑制剂。
3.如权利要求1或2所述的应用,其特征在于,所述的SLC7A11抑制剂包括SLC7A11基因特异性的RNAi、SLC7A11基因特异性的microRNA、SLC7A11基因的shRNA或SLC7A11基因的siRNA。
4.如权利要求3所述的应用,其特征在于,所述的SLC7A11抑制剂为SLC7A11基因的shRNA,所述shRNA的序列为TTCTCCGAACGTGTCA CGTTTC。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110967726.2A CN113679735A (zh) | 2021-08-23 | 2021-08-23 | Slc7a11基因在肝细胞癌介入栓塞术后中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110967726.2A CN113679735A (zh) | 2021-08-23 | 2021-08-23 | Slc7a11基因在肝细胞癌介入栓塞术后中的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113679735A true CN113679735A (zh) | 2021-11-23 |
Family
ID=78581448
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110967726.2A Pending CN113679735A (zh) | 2021-08-23 | 2021-08-23 | Slc7a11基因在肝细胞癌介入栓塞术后中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113679735A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114933646A (zh) * | 2022-06-22 | 2022-08-23 | 南通市第一老年病医院(上海大学附属南通医院、南通市第六人民医院、南通市肺科医院) | Smagp蛋白多肽构建及其抗脂肪性肝病活性的应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104258377A (zh) * | 2014-09-10 | 2015-01-07 | 中南大学湘雅医院 | Pik3c2a蛋白在治疗肝癌药物中的应用 |
CN107998396A (zh) * | 2016-11-02 | 2018-05-08 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 一种肿瘤治疗的靶点及其应用 |
CN108014327A (zh) * | 2016-11-02 | 2018-05-11 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 针对肿瘤相关巨噬细胞的肿瘤免疫治疗靶标 |
CN111996256A (zh) * | 2020-09-04 | 2020-11-27 | 上海市胸科医院 | Slc7a11/ythdc2调控轴在制备治疗肺腺癌的药物中的应用 |
-
2021
- 2021-08-23 CN CN202110967726.2A patent/CN113679735A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104258377A (zh) * | 2014-09-10 | 2015-01-07 | 中南大学湘雅医院 | Pik3c2a蛋白在治疗肝癌药物中的应用 |
CN107998396A (zh) * | 2016-11-02 | 2018-05-08 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 一种肿瘤治疗的靶点及其应用 |
CN108014327A (zh) * | 2016-11-02 | 2018-05-11 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | 针对肿瘤相关巨噬细胞的肿瘤免疫治疗靶标 |
CN111996256A (zh) * | 2020-09-04 | 2020-11-27 | 上海市胸科医院 | Slc7a11/ythdc2调控轴在制备治疗肺腺癌的药物中的应用 |
Non-Patent Citations (3)
Title |
---|
CHANYUAN JIN等: "Inhibition of SLC7A11 by Sulfasalazine Enhances Osteogenic Differentiation of Mesenchymal Stem Cells by Modulating BMP2/4 Expression and Suppresses Bone Loss in Ovariectomized Mice" * |
GUANGSHENG YANG等: "circ-BIRC6, a circular RNA, promotes hepatocellular carcinoma progression by targeting the miR-3918/Bcl2 axis" * |
YUEMING SHEN等: "LincRNA-p21 knockdown enhances radiosensitivity of hypoxic tumor cells by reducing autophagy through HIF-1/Akt/mTOR/P70S6K pathway" * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114933646A (zh) * | 2022-06-22 | 2022-08-23 | 南通市第一老年病医院(上海大学附属南通医院、南通市第六人民医院、南通市肺科医院) | Smagp蛋白多肽构建及其抗脂肪性肝病活性的应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhou et al. | ZEB1 enhances Warburg effect to facilitate tumorigenesis and metastasis of HCC by transcriptionally activating PFKM | |
Liang et al. | Exosomal microRNA-144 from bone marrow-derived mesenchymal stem cells inhibits the progression of non-small cell lung cancer by targeting CCNE1 and CCNE2 | |
Chen et al. | MiR-373 drives the epithelial-to-mesenchymal transition and metastasis via the miR-373-TXNIP-HIF1α-TWIST signaling axis in breast cancer | |
Fang et al. | MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression | |
Huang et al. | miR-340 suppresses glioblastoma multiforme | |
Rong et al. | Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis | |
Chu et al. | MiR-495 regulates proliferation and migration in NSCLC by targeting MTA3 | |
Jing et al. | Long noncoding RNA CRNDE promotes non-small cell lung cancer progression via sponging microRNA-338-3p | |
Yeasmin et al. | Biological and clinical significance of NAC1 expression in cervical carcinomas: a comparative study between squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas | |
Wu et al. | Oncogene FOXK1 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition | |
Yang et al. | MicroRNA‐145 induces the senescence of activated hepatic stellate cells through the activation of p53 pathway by ZEB2 | |
Xu et al. | miR-889 promotes proliferation of esophageal squamous cell carcinomas through DAB2IP | |
Ji et al. | Long non-coding RNA DSCAM-AS1 accelerates the progression of hepatocellular carcinoma via sponging miR-338-3p | |
Zhao et al. | LncRNA TDRG1 functions as an oncogene in cervical cancer through sponging miR-330-5p to modulate ELK1 expression | |
Xu et al. | SNORD47, a box C/D snoRNA, suppresses tumorigenesis in glioblastoma | |
Zhou et al. | LncRNA SNHG16 promotes epithelial-mesenchymal transition via down-regulation of DKK3 in gastric cancer | |
Liu et al. | Krüppel-like factor 8 involved in hypoxia promotes the invasion and metastasis of gastric cancer via epithelial to mesenchymal transition | |
Zhang et al. | Downregulated miR-621 promotes cell proliferation via targeting CAPRIN1 in hepatocellular carcinoma | |
Fu et al. | MicroRNA 27b promotes cardiac fibrosis by targeting the FBW7/Snail pathway | |
Li et al. | Down‐regulation of pescadillo inhibits proliferation and tumorigenicity of breast cancer cells | |
Zhou et al. | MicroRNA-206 attenuates glioma cell proliferation, migration, and invasion by blocking the WNT/β-catenin pathway via direct targeting of Frizzled 7 mRNA | |
Yan et al. | LncRNA DHRS4-AS1 inhibits the stemness of NSCLC cells by sponging miR-224-3p and upregulating TP53 and TET1 | |
Chen et al. | LncRNA SNHG6 promotes G1/S-phase transition in hepatocellular carcinoma by impairing miR-204-5p-mediated inhibition of E2F1 | |
Li et al. | DDX11-AS1exacerbates bladder cancer progression by enhancing CDK6 expression via suppressing miR-499b-5p | |
Sha et al. | BYSL promotes glioblastoma cell migration, invasion, and mesenchymal transition through the GSK-3β/β-catenin signaling pathway |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211123 |