Effect Of Circadian Clock Gene BMAL1 On Regulating Cell Cycle Related Proteins Of Cne1 After Radiotherapy By ATM/ATR Pathway

Objective: Constructed CNE1 Cell line with different expression states of Bmal1 gene to investigate the specific mechanism of Bmal1 gene regulating the expression of cell cycle related proteins through ATM/ATR pathway at the molecular level by comparing changes of related proteins expression before and after radiotherapy.Methods:Constructed CNE1 cell line,Bmal1 gene overexpression group was constructed by lentivirus vector transfection,control group was constructed by empty vector transfection,inhibitor group was constructed by adding KU55933,an inhibitor of ATM/ATR pathway,to the overexpression group,untreated CNE1 Cell was used as blank group.Co-immunoprecipitation(co-IP)was used to detect the interaction between Bmal1 gene and ATM/ATR checkpoints.Using Western blot(WB)to detect the overexpression of Bmal1 gene at protein level and the expression of ATM/ATR pathway related proteins ATM,ATR,chk1,chk2,p-chk1,p-chk2,p53,p21,C-DC25 A,CDC25C,WEEL,Cyclin B1,Cyclin D1,Cyclin E1,CDK1,CDK2,CDK4,CDK6.Results:WB showed Bmal1 gene expression in the overexpression group was higher than blank group and control group(P<0.05),the expression of Bmal1 gene had no significant difference between the blank group and the control group(P>0.05).Co-IP confirmed the interaction between Bmal1 gene and ATM,ATR,chk1,chk2,the chechkpoints of ATM/ATR patyway before and after radiotherapy.The protein expression of ATM,ATR,chk1,p-chk1,p-chk2 and p53,upstream genes of ATM/ATR pathway,in the overexpression group was significantly higher than the blank group and control group before radiotherapy(P<0.05),there was no significant difference in the expression of chk2 gene among these three groups(P > 0.05);the protein expression of P53,P21,p-chk1,p-chk2p53,p21,p-chk1 and p-chk2,the upstreamgenes of ATM/ATR pathway,increased significantly,while the expression of CDC25 A,CDC25C,Cyclin B1,Cyclin D1,Cyclin E1,CDK1,CDK2,CDK4,CDK6 and WEEL,the downstream genes of ATM/ATR pathway,decreased significantly after radiotherapy(P<0.05).Comparing with the overexpression group and inhibitor group showed when the ATM/ATR pathway blocked by inhibitor KU55933,the expression of ATM/ATR pathway upstream genes p53,p21,p-chk1 decreased,and the expression of downstream genes CDC25 A,CDC25C,Cyclin B1,Cyclin D1,Cyclin E1,CDK1,CDK-2,CDK4,CDK6 increased,these differences were statistically significant(P<0.05),while there was no significant difference in the expression of p-chk2,WEEL between these two groups(P>0.05).The expression of p-chk1 in blank group,control group and overexpression group after radiotherapy was significantly higher than before(P<0.05),but there was no significant difference in the expression of p53 and p-chk2 between the three groups before and after radiotherapy(P>0.05).Conclusion:Bmal1 gene could up-regulate the expression of P53 to inhibit cell proliferation.The response of ATM/ATR pathway could be activated after radiotherapy,the overexpression of Bmal1 gene could up-regulate the expression of ATM/ATR pathway upstream genes such as p53,P21,p-chk1 and p-chk2,then inhibited the activity of downstream Cyclin-CDK complexes by phosphorylated CDC25 A and CDC25 C,finally achieved the function of blocking cell cycle after radiotherapy.