The Correlation Study of Circadian Clock Gene BMAL1 Regulates the Biological Behavior of Human Nasopharyngeal Carcinoma Cell After Radiotherapy

Abstract

To investigate the effect of circadian clock gene BMAL1 on proliferation and cell cycle of Nasopharyngeal Carcinoma(NPC) CNE1 cells with different expression status, compare the expression level of pathway related proteins before and after radiotherapy, to further understand the mechanism of BMAL1 gene regulating the biological behavior of NPC cells, aim to explore the mechanism of BMAL1 gene regulating the expression of cell cycle related proteins through ATM/ATR pathway at the molecular level.Constructed CNE1 cell lines with BMAL1 gene overexpression group, control group, inhibitor group, blank group. Using Western blot (WB) to detect the overexpression of BMAL1 gene at protein level and the expression of ATM/ATR pathway related proteins. The absorbance of cells with the same number of plates was determined by a cell counting kit for 6 consecutive days. The effect of BMAL1 gene on the proliferation rate of CNE1 was analyzed. The effect of BMAL1 gene on cell cycle of CNE1 was studied by flow cytometry. Co-immunoprecipitation (co-IP) was used to detect the interaction between BMAL1 gene and ATM/ATR checkpoints.WB showed BMAL1 gene had a higher expression in the overexpression group. In the BMAL1 gene overexpression group, the absorbance values of CNE1 cells were lower at 1 to 6 days, the proportion of G0/G1 phase cells was higher, the relative protein expression of P21,P27 were higher. Co-IP confirmed the interaction between BMAL1 gene and the checkpoints of ATM/ATR pathway before and after radiotherapy. The protein expression of upstream genes of ATM/ATR pathway in the overexpression group was significantly higher before radiotherapy (P < 0.05). The protein expression of the upstream genes of ATM/ATR pathway increased significantly, while the expression of the downstream genes decreased significantly after radiotherapy. 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 decreased, and the expression of downstream genes increased. The expression of p-chk1 in blank group, control group and overexpression group after radiotherapy was significantly higher than before, all the difference were statistically significant.The circadian clock gene BMAL1 inhibits the proliferation of NPC cell line CNE1, arrests cell cycle in the G0/G1 phase by up-regulate the expression of P21,P27 and P53.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 p-chk1 and p-chk2,then inhibited the activity of downstream Cyclin-CDK complexes by phosphorylated CDC25A and CDC25C,finally achieved the function of blocking cell cycle after radiotherapy.