PERK参与调控砷化物诱导细胞自噬反应的信号转导机制研究

Mechanisms involved in PERK-dependent autophagy under arsenite exposure

目的探索PERK是否参与调控砷化物诱导的细胞自噬反应。方法体外培养人肝癌细胞HepG2,以砷化物为刺激源,采用Western印迹方法检测自噬反应标志性蛋白的表达水平、PERK诱导活化状态以及敲低PERK表达水平后砷化物诱导细胞自噬反应和p53诱导活化水平变化情况;采用双荧光素酶报告基因技术检测敲低PERK表达水平后p53的转录激活活性变化。结果砷化物刺激HepG2细胞后自噬反应相关蛋白Beclin-1诱导表达、LC3发生剪切、p62发生降解反应,同时PERK活化水平显著增强;敲低PERK表达水平后,砷化物刺激作用下Beclin-1的诱导表达、LC3的剪切以及p62的降解均被显著抑制;同样条件下p53在Ser15和Ser392位的磷酸化修饰反应水平和转录激活活性显著下降、p53下游靶基因DAPK1的诱导表达水平被显著抑制。结论 PERK能通过调节p53活化及其下游靶基因DAPK1表达从而介导砷化物诱导的细胞自噬反应。

Objective To explore whether PERK is involved in the regulation of arsenite-induced autophagy. Methods Human hepatoma cells HepG2 were cultured and treated with arsenite. The expression level of autophagie hallmarks and the activation status of PERK were detected by Western blotting. The transactivation of p53 and the induction of its downstream target genes expression were also detected by Western blotting after knockdown of PERK expression. Transaetivity of p53 was detected by dual luciferase reporter assay after knockdown of PERK expression. Results An increase in the LC3BII： I ratio, the induction of Beclin-1 expression and the degradation of p62 were readily observed in arsenite-treated HepG2 cells,but the effects were abolished after knockdown of PERK expression. Furthermore, phosphorylation of p53 at Ser15 and Ser392, transactivation of p53 and the induction of its downstream target gene DAPK1 expression were effectively inhibited under the same PERK knockdown conditions. Conclusion PERK regulates arsenite-induced autophagy by activating p53-dependent DAPK1 upregulation.