二甲双胍对人肝癌细胞MHCC97H增殖、凋亡的影响及机制

Effects of metformin on proliferation and apoptosis of human liver cancer cells MHCC97H

目的探究二甲双胍对人肝癌细胞增殖、凋亡的影响及机制。方法将对数生长期的人肝癌MHCC97H细胞分为对照组及实验组1、2、3组和抑制剂组,对照组不予干预,实验1、2、3组分别给予2.5、5、10 mmol/L二甲双胍,抑制剂组给予10 mmol/L二甲双胍+10μmol/L转化生长因子-β_(1)(TGF-β_(1))/Smads通路抑制剂LY2109761。用倒置显微镜、活细胞计数(CCK-8)、5-乙炔基-2´脱氧尿嘧啶核苷(EdU)、Hoechst 33258染色及蛋白免疫印迹(WB)法对细胞形态、增殖活力、增殖率、凋亡情况及相关蛋白表达进行分析。结果与对照组比较,实验1、2、3组的细胞活力呈剂量依赖性降低(P均<0.05),其中实验3组细胞活力接近50%。与对照组相比,实验1、2、3组和抑制剂组MHCC97H细胞生长受到抑制,细胞增殖率、细胞周期蛋白D1、TGF-β_(1)及p-Smad3蛋白表达降低,而细胞凋亡数量、半胱氨酸蛋白酶-3、Smad7蛋白表达增加,且浓度越高变化越显著(P均<0.05),与实验3组相比,抑制剂组各指标变化更显著(P均<0.05)。结论二甲双胍可抑制人肝癌MHCC97H细胞增殖并促进其凋亡,其作用机制可能与抑制TGF-β_(1)/Smads信号通路转导有关。

Objective To investigate the effects of metformin on proliferation,apoptosis,and transforming growth factor-β_(1)(TGF-β_(1))/Smads signaling pathway of human liver cancer cells and the mechanism of action.Methods Human liver cancer MHCC97H cells in the logarithmic growth phase were divided into the control group,experimental groups 1,2,3 and inhibitor group.Cells in the experimental groups 1,2 and 3 were treated with 2.5,5 and 10 mmol/L metformin,respectively.Cells in the inhibitor group were treated with 10 mmol/L metformin+10μmol/L TGF-β_(1)/Smads pathway inhibitor LY2109761.The inverted microscope,cell counting kit-8(CCK-8),5-acetylene-2´deoxyuracil riboside(EdU)staining,Hoechst 33258 staining,and Western blotting were used to detect cell morphology,viability,proliferation,apoptosis and related protein levels.Results Compared with the control group,the cell viability of the experimental groups 1,2 and 3 decreased significantly in a dose-dependent manner(P<0.05),the cell viability of the experimental group 3 was close to 50%.Compared with the control group,the growth of MHCC97H cells in the experimental groups 1,2 and 3 and inhibitor group was inhibited,cell proliferation rate and the protein levels of Cyclin D1,TGF-β1 and p-Smad3 decreased,while the apoptosis and Caspase-3,and Smad7 protein level increased,and the higher the concentration,the more significant the change(P<0.05).Compared with the experimental group 3,the changes of each index in the inhibitor group were more significant(all P<0.05).Conclusion Metformin can significantly inhibit the proliferation and promote apoptosis of MHCC97H cells,which may be related to the inhibition of transduction of TGF-β_(1)/Smads signaling pathway.