二甲双胍通过TGF-β1/Smads通路抑制肝癌细胞的增殖和迁移

Metformin Inhibits Proliferation and Migration of Liver Cancer Cells Through TGF-β1/Smads Pathway

为探究二甲双胍对人肝癌细胞的增殖、迁移及TGF-β1/Smads信号通路的调控作用,体外培养人肝癌MHCC97H细胞,分为对照组(不做干预)、1.25、2.50、5.00 mmol/L二甲双胍组和抑制剂组(5.00 mmol/L二甲双胍+10μmol/L LY2109761)。本研究中5.00 mmol/L二甲双胍组细胞活力接近50%,故选其为最佳浓度。用细胞计数试剂盒-8(CCK-8)、5-乙炔基-2'脱氧尿嘧啶核苷(EdU)、Transwell小室、实时荧光定量PCR(RT-qPCR)及蛋白免疫印迹法对人肝癌MHCC97H细胞的增殖活力、增殖率、迁移能力及相关因子的表达水平进行分析。与对照组相比,2.50、5.00 mmol/L二甲双胍组和抑制剂组细胞的增殖活力、增殖率减少(P<0.05)。1.25、2.50、5.00 mmol/L二甲双胍组和抑制剂组细胞的迁移数、TGF-β1和p-Smad3蛋白的表达水平、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)及纤维黏连蛋白(FN)的mRNA与蛋白质的表达水平较对照组减少,而p-Smad7蛋白的表达水平、E-钙黏蛋白(E-cadherin)的mRNA与蛋白质的表达水平显著增加,且浓度越高变化越显著(P<0.05);与5.00 mmol/L二甲双胍组相比,抑制剂组抑制剂的加入使各指标变化更显著(P<0.05)。二甲双胍可抑制人肝癌MHCC97H细胞的增殖、迁移和上皮间质转化(EMT)依赖于对TGF-β1/Smads通路的抑制作用,这为肝癌的研究提供了参考依据。

In order to explore the regulatory effects of metformin on the proliferation,migration and TGF-β1/Smads signaling pathway of human liver cancer cells,human liver cancer MHCC97H cells were cultured in vitro.They were divided into control group(no intervention),1.25,2.50,5.00 mmol/L metformin group and inhibitor group(5.00 mmol/L metformin+10μmol/L LY2109761).In this study,the cell viability of 5.00 mmol/L metformin group was close to 50%,hence 5.00 mmol/L metformin was selected as the optimal concentration.Cell counting kit-8(CCK-8),5-acetylidene-2'deoxyuracil riboside(EdU),Transwell chamber,real-time fluorescence quantitative PCR(RT-qPCR)and Western blot were used to analyze the proliferation activity,proliferation rate,migration ability and expression levels of related factors of human hepatocellular carcinoma MHCC97H cells.Compared with the control group,the proliferation activity and proliferation rate of 2.50 and 5.00 mmol/L metformin groups and inhibitor groups decreased(P<0.05).The cell migration number,TGF-β1 and p-Smad3 protein expression levels,N-cadherin,vimentin and fibronectin(FN)mRNA and protein expression levels of 1.25,2.50 and 5.00 mmol/L metformin groups and inhibitor group decreased compared with the control group.The expression level of p-Smad7 protein,E-cadherin mRNA and protein significantly increased,and the changes were more significant with the increase of concentration(P<0.05).Compared with 5.00 mmol/L metformin group,the addition of inhibitors in the inhibitor group made the changes of all indexes more significant(P<0.05).The inhibition of metformin on the proliferation,migration and epithelial-mesenchymal transformation(EMT)of MHCC97H cells of human liver cancer depends on the inhibition of TGF-β1/Smads pathway,which provides a reference for the study of liver cancer.