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
发布时间：2019-02-15 01:36
【摘要】：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。
[Abstract]:Objective to investigate whether PERK is involved in the regulation of autophagy induced by arsenide. Methods in vitro cultured human hepatoma cell line HepG2, was stimulated by arsenide and the expression level of autophagy iconic protein was detected by Western blotting. The activation state induced by PERK and the changes of autophagy induced by arsenide and activation induced by p53 after knocking down the level of PERK expression; Double luciferase reporter gene technique was used to detect the transcriptional activation activity of p53 after knockdown of PERK expression level. Results the expression of autophagy associated protein Beclin-1 was induced by arsenide in HepG2 cells, LC3 was shearing, p62 was degraded, and the level of PERK activation was significantly increased. After knocking down the expression level of PERK, the expression of Beclin-1, the shearing of LC3 and the degradation of p62 were significantly inhibited by arsenide stimulation. Under the same conditions, the phosphorylation of p53 at Ser15 and Ser392 sites and the activity of transcriptional activation decreased significantly, while the induced expression of p53 downstream target gene DAPK1 was significantly inhibited. Conclusion PERK can mediate autophagy induced by arsenide by regulating p53 activation and downstream target gene DAPK1 expression.
【作者单位】： 军事医学科学院基础医学研究所军事应激医学研究室;广西医科大学;
【基金】：国家自然科学基金资助项目(31270797,31570758)
【分类号】：R329.2
本文编号：2422825
[Abstract]:Objective to investigate whether PERK is involved in the regulation of autophagy induced by arsenide. Methods in vitro cultured human hepatoma cell line HepG2, was stimulated by arsenide and the expression level of autophagy iconic protein was detected by Western blotting. The activation state induced by PERK and the changes of autophagy induced by arsenide and activation induced by p53 after knocking down the level of PERK expression; Double luciferase reporter gene technique was used to detect the transcriptional activation activity of p53 after knockdown of PERK expression level. Results the expression of autophagy associated protein Beclin-1 was induced by arsenide in HepG2 cells, LC3 was shearing, p62 was degraded, and the level of PERK activation was significantly increased. After knocking down the expression level of PERK, the expression of Beclin-1, the shearing of LC3 and the degradation of p62 were significantly inhibited by arsenide stimulation. Under the same conditions, the phosphorylation of p53 at Ser15 and Ser392 sites and the activity of transcriptional activation decreased significantly, while the induced expression of p53 downstream target gene DAPK1 was significantly inhibited. Conclusion PERK can mediate autophagy induced by arsenide by regulating p53 activation and downstream target gene DAPK1 expression.
【作者单位】： 军事医学科学院基础医学研究所军事应激医学研究室;广西医科大学;
【基金】：国家自然科学基金资助项目(31270797,31570758)
【分类号】：R329.2
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