Histone Deacetylase SIRT1 Confers Resistance To TKI In Non-Small Cell Lung Cancer

Objective:To explore the production of NSCLC resistant cells is related to the increased expression of SIRT1 in CSCs subpopulations,and to study the acquired EGFR-TKI resistance mechanism,and provide theoretical support for exploring strategies to overcome EGFR-TKI resistance.Methods:1.The cell line of drug-resistant strains was successfully established by stepwise induction method,and the ratio of CSCs in drug-resistant cell lines was detected by Aldeflour compared with the parental strain.2.Morphological changes of drug-resistant and parental strains were observed using an inverted microscope.3.The establishment of PC-9-GR resistant strains was detected by CCK8 method,and its IC₅₀ was calculated.4.The Aldeflour?experiment was used to detect the difference in the percentage of ALDH bright CSCs between PC-9 and PC-9-GR.5.Detection of SIRT1 protein expression by WB.6.Detection of SIRT1 mRNA expression by QPCR and quantitative analysis.7.Using cell suspension culture to clone into the ball to detect SIRT1 small molecule inhibitor TV6 reverses the PC-9-GR acquired drug resistance phenotype.8.In vivo,the effect of different treatments on the tumor of nude mice.9.Using SPSS 23.0 for data statistical processing,the results generally use t test.GraphPad Prism5.0 statistical drawing software for data processing,analysis and drawing graphics.Statistical significance was found at p<0.05.Results:1.The drug-resistant cell line PC-9-GR was successfully established,and the induced drug-resistant cells were cultured in a medium containing 1?mol/L of gefitinib.The inverted microscope showed that the drug-resistant strains showed different forms from the parental cell lines,mostly irregular,with large nuclei and increased pseudopods.2.The IC50 of the two cells by CCK8 method showed that the IC50 of PC-9-GR?resistant strain?was significantly higher than that of PC-9?parent strain?.3.The results of the Aldeflour?assay indicated that the proportion of PC-9-GR?resistant strain?stem cells was significantly higher than that of PC-9?parent strain?.In the presence of verapamil?ALDH inhibitor?,most of the ALDH bright in PC-9-GR was eliminated,indicating the specificity of ALDH bright?p=0.0014<0.05?.4.The expression of SIRT1 protein was detected by Western Blot.The results showed that the expression level of SIRT1 protein in PC-9-GR was significantly higher than that of PC-9?p=0.0004<0.05?.5.Detection of SIRT1 mRNA expression by QPCR and quantitative analysis showed that SIRT1 mRNA in PC-9-GR?resistant strain?was significantly higher than PC-9?parental strain??p=0.001<0.05?.6.The stem cell characteristics were detected by cloning into a ball assay,and it was found that the ability of 1PC-9-GR?resistant strain?to clone into a ball was significantly higher than that of PC-9?parent strain??p=0.0109<0.05?;2 PC-9?parent strain?significantly reduced the ability to clone into a ball after treatment?p=0.0049<0.05?;3 PC-9-GR?resistant strain?after application of gefitinib,there was no significant difference in ball-forming ability?p=0.6583>0.05?;after the application of SIRT1 specific inhibitor TV6,the ability to form a ball was significantly reduced?p=0.0006<0.05?;while PC-9-GR?resistant strain?double After the combination of drugs,the ability to form a ball was significantly reduced?p=0.0003<0.05?.7.The effects of different treatments on the tumors of nude mice found that:?1?there was no significant difference in tumor size after gefitinib was applied;?2?the tumor became smaller after application of SIRT1 specific inhibitor TV6;?3?double The tumor became significantly smaller after the combination of the drugs.Conclusion:1.In the NSCLC cell line,the proportion of CSCs and the expression of SIRT1 in the resistant cell lines were higher than those of the parental cells.2.Synergistic administration of gefitinib+TV6 group can more effectively reverse the dry state of acquired drug-resistant cell lines.