CHOP双重调控衣霉素诱导的DU-145细胞凋亡及自噬的研究

Dural regulation of CHOP on apoptosis and autophagy on DU-145 cells induced by tunicamycin

目的研究内质网应激分子CHOP调控细胞凋亡与自噬的作用和机制。方法利用衣霉素诱导DU-145细胞产生内质网应激,Western Blot法检测内质网应激相关分子Grp78、Grp94、p-eIF2α和CHOP及自噬蛋白LC3Ⅱ、Atg5和Beclin1的表达;用流式细胞术检测细胞凋亡水平;沉默CHOP基因,用Western Blot法检测凋亡蛋白PARP、Caspase3的表达,流式细胞术检测细胞凋亡;并利用免疫荧光检测自噬标志性蛋白LC3B的表达。结果衣霉素诱导DU-145细胞内质网应激能诱导一定程度的细胞凋亡,衣霉素处理8、12、24 h的细胞凋亡率分别为3.27%±1.02%,8.97%±0.71%和11.67%±1.41%,处理12 h及24 h的细胞凋亡率与对照组相比差异具有统计学意义(P <0.01)。同时也能通过抑制PI3K/AKt/mTOR信号通路激活DU-145细胞自噬。CHOP基因沉默抑制细胞凋亡,shCtrl组细胞凋亡率为32.17%±3.93%,shCHOP-1组细胞凋亡率为23.53%±3.41%,两组相比差异具有统计学意义(P <0.05)。且CHOP基因沉默能促进细胞自噬分子LC3B的表达。结论衣霉素诱导DU-145细胞内质网应激状态下,CHOP在细胞凋亡与自噬之间发挥双重调控作用。

Objective To explore the role and mechanism of endoplasmic reticulum stress key molecule CHOP in regulating apoptosis and autophagy.Methods Endoplasmic reticulum stress was induced by tunicamycin in DU-145 cells.We detected the expression of endoplasmic reticulum stress-related proteins Grp78,Grp94,p-eIF2α,CHOP and autophagic proteins LC3Ⅱ,Atg5 and Beclin1 by Western Blot,and then we analysed the level of apotosis by flow cytometry.We detected the expression of PARP and Caspase3 by Western Blot and the rate of apotosis by flow cytometry after silencing of CHOP.The expression of autophagic marker LC3B was detected by immunofluorescence assay after silencing of CHOP.Results Tunicamycin could slightly induce apotosis,the apoptotic rate of DU-145 cells treated with tunicamycin for 8,12 and 24 h were 3.27﹪±1.02﹪,8.97﹪±0.71﹪and 11.67﹪±1.41﹪reepectively.The apoptotic rates at 12 h and 24 h were higher than the control group(P<0.01).Tunicamycin could active autophagy in DU-145 cells under endoplasmic reticulum stress through inhibiting PI3K/AKt/mTOR pathway.CHOP gene silencing could inhibit apotosis and promote the expression of LC3B significantly.The apoptosis rates of the shCtrl group and shCHOP group were 32.17﹪±3.93﹪and 23.53﹪±3.41﹪,respectively.There was statistical significance between the two groups(P<0.05).Conclusions CHOP palys a dural regulatory role between apoptosis and autophagy in DU-145 cells under endoplasmic reticulum stress induced by tunicamycin.