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NS3/4A是丙型肝炎病毒(hepatitis C virus,HCV)编码的丝氨酸蛋白酶复合体,是病毒完成自身复制周期的必要成分。该研究为调查NS3/4A对细胞凋亡及DNA损伤应答(DNA-damage response,DDR)的影响,在Huh7细胞中表达了外来NS3/4A基因。通过DAPI染色和MTT分析显示,外来表达NS3/4A显著诱导细胞的凋亡和增殖活力的下降。免疫荧光检测结果表明,NS3/4A可明显增加细胞内源性DNA双链断裂(double strand breaks,DSBs)损伤(γH2AX灶点升高);而进一步用X-ray诱导细胞外源性DSBs损伤后,外来表达NS3/4A的细胞显示出明显的DSBs损伤修复缺陷(减缓的γH2AX灶点消退)。免疫印迹法检测结果显示,NS3/4A可抑制喜树碱(Camptothecin,CPT)诱导的ATM第1 981位丝氨酸的磷酸化(pATM1 981)。以上结果提示,NS3/4A基因外来表达可引起细胞DNA损伤,抑制ATM介导的DSBs损伤修复信号,诱导细胞凋亡通路的活化。
NS3 / 4A is a serine protease complex encoded by the hepatitis C virus (HCV), which is an essential component of the virus to complete its own replication cycle. In order to investigate the effect of NS3 / 4A on apoptosis and DNA-damage response (DDR), this study expresses the NS3 / 4A gene in Huh7 cells. DAPI staining and MTT analysis showed that extrinsic expression of NS3 / 4A significantly induced apoptosis and decreased viability of cells. The result of immunofluorescence showed that NS3 / 4A could obviously increase the damage of endogenous double strand breaks (DSBs) in cells (γH2AX foci were elevated), while X-ray induced the injury of exogenous DSBs , Cells exogenously expressing NS3 / 4A showed a marked defect in DSBs damage repair (slowing down of the [gamma] H2AX foci). Western blotting showed that NS3 / 4A inhibited the serine phosphorylation of ATM at position 1 981 induced by camptothecin (CPT) (pATM1 981). The above results suggest that exogenous expression of NS3 / 4A gene can cause DNA damage in cells, inhibit ATM-mediated DSBs injury repair signal, and induce activation of apoptosis pathway.