摘要
目的:探讨PERK-EIF2α通路在丹参二萜醌诱导肺癌PC9细胞凋亡中的作用。方法:采用0、2.5、5.0、7.5μg/mL丹参二萜醌处理PC9细胞24h后,CCK8法、Annexin V-FITC/PI双染色法分析细胞增殖活性及凋亡情况;Western blot检测凋亡相关蛋白Bax、Bcl-2,PERK-EIF2α通路相关关键蛋白,以及下游CHOP、ATF4蛋白表达水平;PERKsi RNA转染PC9细胞,Western blot检测PERK蛋白的转染结果,筛选出干扰效果最佳的片段。Annexin V-FITC/PI双染色法检测沉默PERK基因后丹参二萜醌对PC9细胞的凋亡情况,Western blot分析PERK-EIF2α通路相关关键蛋白的表达情况。结果:随着丹参二萜醌浓度的增加,PC9细胞增殖抑制作用增加,凋亡率显著增高(P<0.05);随着时间延长PERK、EIF2α蛋白磷酸化水平明显增加,且以8h增加最为明显(P<0.05);其下游蛋白ATF4表达量逐渐减低,CHOP逐渐增高,呈剂量依赖性。PERK蛋白干扰后研究得到与干扰前相反的结果:与空载体组比较,丹参二萜醌诱导PC9细胞凋亡被明显抑制(P<0.05);PERK、EIF2α蛋白磷酸化水平明显降低(P<0.05),ATF4表达量有所增加,CHOP有所降低。结论:丹参二萜醌可触发PC9细胞内质网应激,其诱导细胞凋亡的机制与激活PERK/EIF2α通路密切相关。
Objective: To investigate the effect of PERK-EIF2α pathway on the apoptosis of lung cancer PC9 cell induced by diterpenoid tanshinonesand. Methods: PC9 cells were treated with 0, 2.5, 5.0, 7.5μg/mL diterpenoid tanshinonesand respectively for 24 hours.The cell proliferation and apoptosis were analyzed by CCK8 assay and Annexin V-FITC/PI. The expressions of apoptosis-related proteins Bax and Bcl-2, PERK-EIF2αpathway related key protein, and downstream protein CHOP and ATF4 were detected by Western blot. The RNA interference fragment targeting to PERK gene was transfected into PC9 cells, and the expression of PERK si RNA was detected by Western blot to select the best interference fragment.Apoptosis of PC9 cell induced by diterpenoid tanshinonesand after silencing PERK gene was detected by Annexin V-FITC/PI double staining method.The expression of PERK-EIF2α pathway related key proteinwas analyzed by Western blot. Results: With the increase of diterpenoid tanshinonesand concentration, theinhibitory effect of PC9 cell proliferation increased and the apoptosis rate was significantly increased(P<0.05). With the prolongation of time, the phosphorylation levels of PERK and EIF2α protein were significantly increased,especially for 8h(P<0.05). The expression of downstream protein ATF4 decreased gradually, and the expression of CHOP increased gradually, which are in a dose-dependent manner. Compared with the blank group, the silence of PERK gene could significantly increase the viability of PC9 cell and the inhibit the apoptosis(P<0.05). The phosphorylation levels of PERK and EIF2α protein were significantly decreased(P<0.05), while the expression of downstream protein ATF4 increased, and the expression of CHOP decreased. Conclusion: Diterpenoid tanshinones can induce PC9 cell endoplasmic reticulum stress,and its mechanism of cell apoptosis is closely related to activation of PERK/EIF2α pathway.
引文
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