Effects Of Circadian Clock Gene BMAL1 On Proliferation And Radiosensitivity Of Human Nasopharyngeal Carcinoma Cell CNE2

Objective:To investigate the effects of circadian clock gene BMAL1 on proliferation,apoptosis and radiosensitivity of human nasopharyngeal carcinoma cell CNE2.Methods:Lentvirus vectors with BMAL1 interference and BMAL1 overexpression were constructed and infected into CNE2 and screened by puromycin for two weeks.The gene silence and overexpression efficiency on mRNA and protein level of these recombinants were detected by quantitative real-time PCR and western blot.Cell proliferation and radiation inhibition rate were measured by CCK-8 assay.Apoptosis and cell cycle were detected by flow cytometry.The effect of BMAL1 on radiosensitivity irradiated different dose(0、2、4、6Gy)were measured by colony formation assay.Results:Recombinant lentivirus pGMLV-SB3 shRNA3 inhibited BMAL1 expression at mRNA and protein level,the expression level of mRNA decreased by0.806(P=0.002)and protein level decreased by 0.831(P=0.000);lentivirus pGMLV-PA6-BMAL1 over expressed BMAL1 at mRNA and protein level,the expression level of mRNA increased to 7.191(P=0.003)and protein level increased to 2.503(P=0.031),the difference is statistically significant.The cell proliferation of BMAL1 interference group were faster compared with negative control(P<0.001)and BMAL1 overexpression group were lower compared with negative control(P<0.001),the difference is statistically significant.The absorbance of BMAL1 interference group after irradiation 72 hour was higher(P<0.001)and the radiation inhibition rate was lower compared with control group(P=0.001);the absorbance of BMAL1 overexpression group after irradiation 72 hour was lower(P<0.001)and the radiation inhibition rate was higher compared with control group(P<0.001),the difference is statistically significant.The apoptosis rate of BMAL1 interference group was lower than control group(P=0.007).The apoptosis rate of BMAL1 overexpression group was higher than control group(P=0.004),the difference is statistically significant.After irradiation 24hours of 8Gy X-rays,the S phase of BMAL1 interference group was higher compared with control group(P=0.001)and the S phase of BMAL1 overexpression group was lower(p=0.000);the G2/M phase of BMAL1 interference group was lower compared with control group(P=0.000)and the G2/M phase of BMAL1 overexpression group was higher compared with negative control(P=0.001),the difference is statistically significant.After irradiation 48 and 72hours of 8Gy X-rays,the cell cycle distribution was no statistical difference.The plating efficiency of BMAL1 interference group was higher(P=0.032)and the survival fraction was also higher compared with control group(P=0.036),and its sensitizing enhance rate(SER)was 0.512.The plating efficiency of BMAL1 overexpression group was lower(P=0.042)and the survival fraction was also lower compared with control group(P=0.037),and its sensitizing enhance rate(SER)was 1.794.Conclusioin:circadian clock gene BMAL1 inhibited nasopharyngeal carcinoma cell CNE2 proliferation,increased apoptosis induced by X-radiation and radiosensitivity through reducing the proportion of S phase and regulating G2/M arrest.