Huang Ya Wei, Xiong Li, Dou De Yu, Lyu Meng Juan, Ma Yu Hong
Clinical Medical Experiment Training Center,Wannan Medical College.
Department of Central Lab, Wannan Medical College, Wuhu 241000, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2021 May;37(3):266-271. doi: 10.12047/j.cjap.6039.2021.013.
To investigate the effect of TGF-β1/Smad signaling pathway on the apoptosis of HepG2 cells under endoplasmic reticulum stress (ERS). An ERS model was established firstly. Human hepatocellular carcinoma HepG2 cells were treated with 3 μmol/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 (3 μmol/L TM treatment group), TM + NC group(3 μmol/L TM + si-TGF-β1 negative control group), TM + si-TGF-β1 group(3 μmol/L TM + si-TGF-β1 group), TM + pEX-3 group(3 μmol/L TM + plasmid control group), and TM + TGF-β1 pEX-3 group(3 μmol/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. Compared with the untreated group, the expressions of TGF-β1 and p-Smad2 in TM group were significantly reduced (＜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 (＜ 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 (＜0.01). 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.
探讨内质网应激（ERS）条件下转化生长因子-β1（TGF-β1）/Smad信号通路对人肝癌HepG2细胞凋亡的影响。首先建立ERS模型。将人肝癌HepG2细胞用3 μmol/L衣霉素（TM）处理24 h以诱导ERS。细胞分为6组，每组设3个复孔，实验重复3次。6组分别为未处理组、TM组（3 μmol/L TM处理组）、TM + NC组（3 μmol/L TM + si-TGF-β1阴性对照组）、TM + si-TGF-β1组（3 μmol/L TM + si-TGF-β1组）、TM + pEX-3组（3 μmol/L TM +质粒对照组）和TM + TGF-β1 pEX-3组（3 μmol/L TM + TGF-β1过表达质粒组）。采用脂质体转染法将TGF-β1小干扰RNA（TGF-β1 si-RNA）和TGF-β1过表达质粒（TGF-β1 pEX-3）转染至HepG2细胞。转染24 h后，采用实时定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法（Western blot）检测各组HepG2细胞中TGF-β1和磷酸化Smad2（p-Smad2）的表达。采用细胞计数试剂盒-8（CCK-8）法和流式细胞术分析各组HepG2细胞增殖抑制率和凋亡率的变化。与未处理组比较，TM组TGF-β1和p-Smad2的表达显著降低（＜0.05）。与TM组比较，TM + si-TGF-β1组TGF-β1和p-Smad2的表达、细胞增殖抑制率及凋亡率均明显降低（＜0.01），而TM + TGF-β1 pEX-3组TGF-β1和p-Smad2的表达、细胞增殖抑制率及凋亡率均显著升高（＜0.01）。ERS条件下人肝癌HepG2细胞中TGF-β1/Smad信号通路被抑制，激活该信号通路后，ERS条件下HepG2细胞的凋亡率显著增加。
