PERK參與調(diào)控砷化物誘導細胞自噬反應(yīng)的信號轉(zhuǎn)導機制研究
發(fā)布時間：2019-02-15 01:36
【摘要】：目的探索PERK是否參與調(diào)控砷化物誘導的細胞自噬反應(yīng)。方法體外培養(yǎng)人肝癌細胞HepG2,以砷化物為刺激源,采用Western印跡方法檢測自噬反應(yīng)標志性蛋白的表達水平、PERK誘導活化狀態(tài)以及敲低PERK表達水平后砷化物誘導細胞自噬反應(yīng)和p53誘導活化水平變化情況;采用雙熒光素酶報告基因技術(shù)檢測敲低PERK表達水平后p53的轉(zhuǎn)錄激活活性變化。結(jié)果砷化物刺激HepG2細胞后自噬反應(yīng)相關(guān)蛋白Beclin-1誘導表達、LC3發(fā)生剪切、p62發(fā)生降解反應(yīng),同時PERK活化水平顯著增強;敲低PERK表達水平后,砷化物刺激作用下Beclin-1的誘導表達、LC3的剪切以及p62的降解均被顯著抑制;同樣條件下p53在Ser15和Ser392位的磷酸化修飾反應(yīng)水平和轉(zhuǎn)錄激活活性顯著下降、p53下游靶基因DAPK1的誘導表達水平被顯著抑制。結(jié)論 PERK能通過調(diào)節(jié)p53活化及其下游靶基因DAPK1表達從而介導砷化物誘導的細胞自噬反應(yīng)。
[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.
【作者單位】： 軍事醫(yī)學科學院基礎(chǔ)醫(yī)學研究所軍事應(yīng)激醫(yī)學研究室;廣西醫(yī)科大學;
【基金】：國家自然科學基金資助項目(31270797,31570758)
【分類號】：R329.2
本文編號：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.
【作者單位】： 軍事醫(yī)學科學院基礎(chǔ)醫(yī)學研究所軍事應(yīng)激醫(yī)學研究室;廣西醫(yī)科大學;
【基金】：國家自然科學基金資助項目(31270797,31570758)
【分類號】：R329.2
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1 康煥華;;毒不死的微生物[J];科學之友;1996年04期
，本文編號：2422825
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