The Circadian Gene CRY1 Mediating Autophagy Regulates The Cisplatin Resistance Of Ovarian Cancer Cell A2780/DDP

Objective: To detect the difference of CRY1 and autophagy in ovarian cancer cisplatin-resistant cell line A2780/DDP and its parental cell line A2780,to explore the correlation between CRY1 and autophagy,and to investigate whether CRY1 is involved in A2780/DDP resistance.Methods:1.qRT-PCR and Western Blot were used to detect autophagy and circadian gene and related protein expression levels in A2780 and A2780/DDP.Immunofluorescence was employed to explore the expression and distribution of LC3 and CRY1.2.After cisplatin treatment,CCK8 detected the survival rate of cells for 1~5 days;Acridine orange(AO)and Hoechst33342 observed autophagic vesicles and nuclear morphology;Western Blot detected autophagy and circadian gene expression.3.3-methyl adenine(3MA)was used to treat A2780 and A2780/DDP cell lines,and Western Blot was used to detect the expression changes of autophagy and circadian genes.4.Lentiviral-mediated CRY1 interference and overexpression plasmid regulated the expression of CRY1 in A2780 and A2780/DDP cells respectively;qRT-PCR and Western Blot detected CRY1 stably transfected cells and autophagy-associated proteins;CCK8 detected cisplatin sensitivity changes before and after CRY1 being regulated.Result:1.Compared with A2780 cells,qRT-PCR results showed that CRY1,CRY2,CLOCK and LC3 were up-regulated in A2780/DDP,and p62/SQSTM1 was down-regulated.The results of Western Blot showed that LC3 was up-regulated,and the expressions of p62/SQSTM1,CRY1,CRY2 and CLOCK were significantly down-regulated.The difference of CRY1 and CLOCK between A2780 and A2780/DDP was most significant.The results of Immunofluorescence showed that the expression of LC3 was significantly increased and CRY1 was decreased in A2780/DDP cells.2.A2780 and A2780/DDP were treated with cisplatin.Compared with A2780,CCK8 results showed that A2780/DDP was more resistant to cisplatin,with IC50 of22.131 μg/ml and 25.378 μg/ml for 24 h and 48 h,respectively,and 6.066 μg/ml and4.316 μg/ml for A2780 IC50,respectively.3.After 48 h treatment with cisplatin,AO results showed that cisplatin induced autophagy effects in A2780 and A2780/DDP.Hoechst33342 observed nuclear morphology,and the results showed apoptosis effects of A2780 and A2780/DDP were significantly increased when cisplatin concentrations were 5 μg/ml and 10 μg/ml,respectively.Compared with A2780,Western Blot results showed that when the concentration of cisplatin was not more than 5 μg/ml,the autophagy effect of A2780/DDP cells was not significantly increased,and CRY1 was on the rise.However,when cisplatin concentration reached 20 μg/ml,the autophagy effect of A2780/DDP was significantly increased,and CRY1 was down-regulated.4.After 3-MA inhibited autophagy,Western Blot showed that the expression of CRY1 in A2780 and A2780/DDP cells increased,but the difference between the two was similar.5.The lentiviral-mediated shCRY1 and 3xflag CRY1 recombinant plasmids were stably transfected into A2780 and A2780/DDP cells,respectively.The mRNA and protein levels of CRY1 in the former showed a downward trend,and the latter was significantly up-regulated.Western Blot detected LC3.The results showed that there was no significant difference in autophagy effect after interference with CRY1.After CRY1 overexpression,A2780 had no significant effect on autophagy,and A2780/DDP cells had an increased autophagy effect.CCK8 detected the survival rate of steady-transfected cell line 1 to 5 days after treatment with cisplatin.It was found that overexpression of CRY1 increased autophagy effect,but cell survival rate decreased.However,there was no significant difference in autophagy expression after CRY1down-regulated.However,its survival rate is on the rise.Conclusion:1.The expression of CRY1 of A2780/DDP cells was significantly decreased,and the autophagy effect was significantly increased.Cisplatin can induce an increase inautophagy effect and a decrease in the expression of CRY1.2.Inhibition of autophagy CRY1 expression was significantly increased.CRY1 gene silencing reduced the sensitivity of A2780 and A2780/DDP cells to cisplatin,the former decreasing more significantly.CRY1 overexpression can effectively reverse the resistance of A2780/DDP cells to cisplatin.3.Autophagy may play a role in A2780/DDP cells by down-regulating CRY1 expression,and up-regulation of CRY1 may increase cisplatin sensitivity of A2780/DDP.CRY1 may be a new target for targeted therapy of refractory ovarian cancer.