The Study On The Mechanism Of ATF4 Regulates Autophagy And Reverses Cisplatin Resistance In Ovarian Cancer A2780/DDP Cell Lines

PurposeTo observe the differences in the expression of ATF4,Beclin1,LC3 and other autophagy-related genes in ovarian cancer A2780 cells and its drug-resistant cell line A2780/DDP cells,and explore the role of ATF4 in regulating autophagy to reverse ovarian cancer drug-resistant cell line A2780/DDP cells cisplatin Possible mechanisms of drug resistance.MethodRNA and protein expression of ATF4 and autophagy-related genes(Beclin1,ATG5,LC3,etc.)in ovarian cancer A2780 cells and drug-resistant cells A2780/DDP cell lines were detected by real-time quantitative RT-PCR and Western blot.ATF4 overexpression and CRISPR-Cas9 knockout ATF4 plasmid were constructed.Virus-mediated transfection of the above two plasmids and blank control plasmids into ovarian cancer cisplatin-resistant cell line A2780/DDP cells,respectively,and stable expression strains were selected.RT-PCR,Western blot and other experimental methods were used to detect RNA and protein expression differences of ATF4,CRY1 and autophagy related genes(Beclin1,LC3,etc.)in each transfected cell line;autophagy inhibitor chloroquine diphosphate was used to inhibit autophagy;Then,the changes of ATF4 and autophagy-related genes Beclin1 and LC3 protein were detected;protein immunocoprecipitation was used to verify whether ATF4 and Beclin1 had a direct effect;and CCK8 was used to determine the cisplatin half-inhibition concentration of different cell lines after transfection IC50)and calculate the resistance index(RI).Result1.RT-PCR and Western blot results showed that the expression of ATF4,CRY1 and autophagy-related genes(Beclin1,ATG5,LC3,etc.)were significantly higher in A2780/DDP cells than in A2780 cells,while the expression of CRY1 in A2780 cells was significantly higher than that of A2780/DDP cells.2.Western blot results showed that after ATF4 was knocked out,the protein expression of autophagy-related genes(Beclin1,ATG5,LC3,etc.)in A2780/DDP cells was suppressed,while the expression of CRY1 protein was not affected.After over-expression of ATF4,the protein expressions of ATF4,CRY1 and autophagy-related genes(Beclin1,ATG5,LC3)were slightly up-regulated in A2780/DDP cells,but there was no statistical significance.The protein expression of P62 was down-regulated and had statistical significance..3.After chloroquine diphosphate was applied to A2780/DDP cells knocking out ATF4,the autophagy-related gene LC3 protein accumulated in the cells,and the autophagy was inhibited.The Beclin1 protein was up-regulated,and the expression level of CRY1 was similar to that without chloroquine diphosphate.No significant difference was seen in the cells.4.The protein co-immunoprecipitation experiment suggested that there was a direct correlation between ATF4 and Beclin1 in A2780/DDP cells.5.The results of CCK8 experiment indicated that the resistance of A2780/DDP cells to cisplatin was significantly decreased and the sensitivity was significantly enhanced after ATF4 was knocked out.Conclusion1.Different detection methods,such as RT-PCR and Western blot,have proved that The expression of ATF4,autophagy-related genes(Beclin1,ATG5,LC3,etc.)and CRY1 genes in A2780 cell line is significantly different with A2780/DDP cell line.2.The high positive rate of ATF4 expression in ovarian cancer cisplatin-resistant cell line A2780/DDP is correlated with ovarian cancer cell cisplatin resistance.3.Down-regulation of ATF4 expression can reverse the resistance of ovarian cancer cisplatin-resistant cell line A2780/DDP to cisplatin.Autophagy plays an important role in this process.4.CRY1 may be involved in regulating the expression of ATF4.