调节TGF-β1/Smad信号通路对内质网应激状态下肝癌HepG2细胞凋亡的影响

Effect of regulating TGF-β1/Smad signaling pathway on apoptosis of hepatocellular carcinoma HepG2 cells under endoplasmic reticulum stress

目的:探讨TGF-β1/Smad信号通路对内质网应激(ERS)状态下肝癌HepG2细胞凋亡的影响机制。方法:首先建立内质网应激模型:以3μmol/L的衣霉素(TM)处理人肝癌HepG2细胞株24 h,诱导细胞发生ERS。实验分为6组,每组3个复孔,实验重复3次,6组分别为:Untreated组(未处理组)、TM组(3μmol/L TM处理组)、TM+NC组(3μmol/L TM+si-TGF-β1阴性对照组)、TM+si-TGF-β1组(3μmol/L TM+si-TGF-β1组)、TM+pEX-3组(3μmol/L TM+质粒对照组)及TM+TGF-β1 pEX-3组(3μmol/L TM+TGF-β1过表达质粒组),利用脂质体的方法将TGF-β1小干扰RNA(si-TGF-β1)及TGF-β1过表达质粒(TGF-β1 pEX-3)转染入HepG2细胞,转染24 h后,利用RT-qPCR和Western blot检测各组HepG2细胞TGF-β1/Smad信号通路相关因子TGF-β1、p-Smad2表达的情况;CCK-8和流式细胞术分别检测各组HepG2细胞增殖抑制率和凋亡率变化情况。结果:与Untreated组相比,TM组细胞的TGF-β1及p-Smad2的表达明显降低(P<0.05);与TM组相比,TM+si-TGF-β1组细胞的TGF-β1及p-Smad2的表达和细胞的增殖抑制率、凋亡率显著降低(P<0.01),而TM+TGF-β1 pEX-3组细胞的TGF-β1及p-Smad2的表达和细胞增殖抑制率、凋亡率显著升高(P<0.01)。结论:TGF-β1/Smad信号通路在肝癌HepG2细胞发生ERS后受到抑制,当该通路被激活后,ERS状态下肝癌HepG2细胞的凋亡率显著升高。

Objective:To investigate the effect of TGF-β1/Smad signaling pathway on the apoptosis of HepG2 cells under endoplasmic reticulum stress(ERS).Methods:An ERS model was established firstly.Human hepatocellular carcinoma HepG2 cells were treated with 3μmol/L tunicamycin(TM)for 24 h to induce ERS.Cells were divided into 6 groups,each with 3 replicate holes,and the experiment was repeated 3 times.The 6 groups included untreated group,TM group(3μmol/L TM treatment group),TM+NC group(3μmol/L TM+si-TGF-β1 negative control group),TM+si-TGF-β1 group(3μmol/L TM+si-TGF-β1 group),TM+pEX-3 group(3μmol/L TM+plasmid control group),and TM+TGF-β1 pEX-3 group(3μmol/L TM+TGF-β1 overexpressed plasmid group).HepG2 cells were transfected with TGF-β1 small interfering RNA(TGF-β1 si-RNA)and TGF-β1 overexpressed plasmids(TGF-β1 pEX-3)by Lipofectamine.Twenty-four hours after transfection,RT-qPCR and Western blot were used to detect the expression of TGF-β1 and p-Smad2 in HepG2 cells of each group.CCK-8 and flow cytometry were used to analyze changes in the proliferation inhibition rate and apoptosis rate of HepG2 cells in each group.Results:Compared with the untreated group,the expressions of TGF-β1 and p-Smad2 in TM group were significantly reduced(P<0.05).Compared with the TM group,the expressions of TGF-β1 and p-Smad2,as well as the cell proliferation inhibition rate and apoptosis rate in TM+si-TGF-β1 group were obviously decreased(P<0.01),while the expressions of TGF-β1 and p-Smad2,cell proliferation inhibition rate and apoptosis rate of TM+TGF-β1 pEX-3 group were significantly increased(P<0.01).Conclusion:The TGF-β1/Smad signaling pathway was inhibited in hepatocellular carcinoma HepG2 cells under ERS,when this pathway was activated,the apoptosis rate of HepG2 cells under ERS was increased significantly.