Beclin 1介导NS5ATP9对饥饿诱导的HepG2细胞凋亡的作用

Beclin 1-mediated inhibitory effect of NS5ATP9 on starving-induced apoptosis in HepG2 cells

目的探讨Beclin 1在NS5ATP9抑制饥饿诱导肝母细胞瘤HepG2细胞凋亡中的作用。方法将HepG2细胞用EBSS饥饿不同时间点,通过蛋白质印迹法检测NS5ATP9、Beclin1及bax蛋白的表达变化;将NS5ATP9的siRNA及过表达载体分别转染HepG2细胞48 h后,用EBSS饥饿细胞24 h,通过蛋白质印迹检测bax的变化;应用Beclin 1siRNA抑制Beclin 1表达后,转染NS5ATP9过表达载体,EBSS饥饿细胞24 h,利用化学发光法检测胱冬肽酶-3/7(caspase-3/7)的活性变化,同时用蛋白质印迹法检测NS5ATP9、Beclin1,bax蛋白的表达变化。结果饥饿HepG2细胞0、6、12、18、24 h后,NS5ATP9及bax的蛋白表达量相应升高。NS5ATP9表达被抑制后,bax蛋白表达量增加。饥饿HepG2细胞24 h后,Beclin 1表达被抑制,caspase-3/7的活性增加(P=0.001),NS5ATP9抑制caspase-3/7活性的作用减弱(P<0.01)。NS5ATP9对bax的负性调控作用减弱。结论Beclin 1通过下调bax的表达部分参与了NS5ATP9抑制饥饿诱导HepG2细胞凋亡的作用。

Objective To investigate Beclin 1-mediated inhibitory effect of NS5ATP9 on starving-induced apoptosis in hepatoblastoma cell line HepG2 cells.Methods HepG2 cells were serum starved and collected at different time points.The expression changes of NS5ATP9,Beclin1 and Bax proteins were detected by Western blot.In addition,HepG2 cells were transfected with NS5ATP9 siRNA or its overexpression vector for 48h.The cells were then serum starved for 24 h and collected for detection of bax by Western blot.Moreover,after knockdown of Beclin 1 by siRNA or overexpression of NS5ATP9 by transfected vector,the cells were serum starved for 24 h and collected for the detection of caspase-3/7 activity by chemiluminescence method.The expression of NS5ATP9,Beclin1 and Bax proteins were detected by Western blot.Results(1)the protein expressions of NS5ATP9 and bax in HepG2 cells were increased after 0,6,12,18 and 24 h of serum starvation.The bax protein expression was increased After siRNA knockdown of NS5ATP9 expression.(2)After serum starvation for 24 h,the activity of Caspase-3/7 in HepG2 cells was increased in the Beclin 1 expression-inhibited cells(P=0.001),this was accompanied by a decreased effect of NS5ATP9 on inhibiting caspase-3/7 activity(P=0.000),and a weakened negative regulatory effect of NS5ATP9 on bax.Conclusion Beclin 1 partly mediated the inhibitory effect of NS5ATP9 on starving-induced HepG2 cell apoptosis through down-regulation of bax expression.