DAPK1调控砷化物诱导的细胞自噬反应机制研究

Involvement of DAPK1 in regulating autophagy under arsenite exposure

目的探究死亡相关蛋白激酶1(DAPK1)在砷化物As_(2)O^(3)诱导细胞自噬反应中的作用及分子机制。方法应用As_(2)O^(3)刺激HepG2细胞后,蛋白质免疫印迹和逆转录PCR实验检测DAPK1、自噬标志性信号分子和DNA损伤调节自噬相关蛋白1(DRAM1)的表达水平及p53活化能力;采用双荧光素酶报告分析实验检测敲低DAPK1后p53的转录激活能力变化情况,流式细胞术检测敲低DAPK1对As_(2)O^(3)诱导细胞自噬反应的影响。结果As_(2)O^(3)刺激能诱导HepG2细胞中DAPK1表达;敲低DAPK1表达显著抑制了细胞自噬反应水平;同时p53的诱导活化反应强度显著下调,但自噬反应特异性p53靶基因DRAM1的表达水平无明显变化。此外,敲低p53表达可显著抑制砷化物刺激作用下DAPK1的诱导表达水平。结论DAPK1在As_(2)O^(3)诱导细胞自噬反应中可通过与p53形成正反馈通路实现对自噬信号的放大效应。

Objective To investigate the role and molecular mechanism of death-associated protein kinase 1(DAPK1)in autophagy induced by arsenite As_(2)O^(3).Methods HepG2 cells were stimulated with As_(2)O^(3).The expressions of DAPK1,DNA damage-regulated autophagy modulator protein 1(DRAM1),autophagic markers and the activation of p53 were detected by Western blotting and reverse transcription PCR assays.The transcriptional activity of p53 and autophagy after knockdown of DAPK1 expressions were detected by dual-luciferase report assay and flow cytometric assay,respectively.Results As_(2)O^(3)induced the up-regulation of DAPK1 in HepG2 cells.Knocking down of DAPK1 expressions significantly inhibited autophagy and the transactivation of p53,but DRAM1,the specific p53 target gene for mediating autophagy,remained unchanged.Moreover,DAPK1 expressions induced by As_(2)O^(3)were significantly inhibited after p53 expressions were knocked down.Conclusion DAPK1 can amplify autophagy signals by forming a positive feedback pathway with p53 in As_(2)O^(3)-induced responses.