论文部分内容阅读
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 investigate whether PERK is involved in the regulation of arsenide-induced autophagy. Methods Human hepatocellular carcinoma HepG2 cells were cultured in vitro. The arsenic compounds were used as stimulus. Western blotting was used to detect the expression of autophagy protein, PERK-induced activation status and PERK expression level. Arsenic-induced autophagy and p53 Induced changes in activation level; dual luciferase reporter gene technology was used to detect the transcriptional activation of p53 after knockdown of PERK expression level. RESULTS: Beclin-1 was induced by arsenic-induced autophagic response protein Beclin-1 in HepG2 cells. LC3 was cleaved and p62 was degraded. At the same time, PERK activation level was significantly increased. After knockdown of PERK expression, Beclin-1 , The cleavage of LC3 and the degradation of p62 were significantly inhibited. Under the same conditions, the level of phosphorylation and transcriptional activation of p53 at Ser15 and Ser392 decreased significantly, and the expression level of DAPK1, a downstream target of p53, was significantly increased inhibition. Conclusion PERK can mediate arsenic-induced autophagic reaction by regulating p53 activation and its downstream target gene DAPK1 expression.