二甲双胍对转化生长因子-β_(2)(TGF-β_(2))诱导的人晶状体上皮细胞增殖、迁移及上皮间质转化的影响

Effects of metformin on proliferation,migration and epithelial-mesenchymal transition of human lens epithelial cells induced by transforming growth factor-β_(2)

目的研究二甲双胍对转化生长因子-β_(2)(TGF-β_(2))诱导的人晶状体上皮细胞(LEC)增殖、迁移及上皮间质转化的影响。方法选择永生化人LEC(HLEB-3细胞)作为细胞来源;将细胞融合度80%的人LEC置于含10 mg·L^(-1)TGF-β_(2)的DMEM低糖培养基中培养24 h作为对照组,经TGF-β_(2)处理再加入不同浓度二甲双胍进一步作用后的细胞作为实验组。处理后在倒置显微镜下观察各组细胞的形态变化。采用CCK-8实验检测细胞毒性,计算细胞存活率,Western blot法检测细胞中辅助激活因子Yes相关蛋白1(YAP1)、大肿瘤抑制因子1(LATS1)、波型蛋白(Vimentin)的表达,实时荧光定量PCR检测YAP1、LATS1、哺乳动物STE20样激酶1(MST1)、Vimentin、E-钙黏蛋白mRNA的表达。结果二甲双胍细胞毒性检测结果显示,当二甲双胍浓度大于15.0 mmol·L^(-1)时,人LEC的存活率明显降低,表明二甲双胍浓度对LEC存活影响较大,因此选择15.0 mmol·L^(-1)进行后续实验。二甲双胍对TGF-β_(2)诱导的人LEC增殖有显著的抑制作用,且呈明显的剂量依赖性(均为P<0.001)。15.0 mmol·L^(-1)二甲双胍作用于人LEC 24 h后细胞中YAP1和Vimentin蛋白相对表达量均低于对照组(均为P<0.05);LATS1蛋白相对表达量高于对照组(P<0.05)。15.0 mmol·L^(-1)二甲双胍作用于人LEC 24 h后细胞中YAP1和Vimentin mRNA相对表达量均低于对照组,LATS1、MST1、E-钙黏蛋白mRNA相对表达量均高于对照组,差异均有统计学意义(均为P<0.05)。结论二甲双胍在体外能够对TGF-β_(2)诱导的人LEC增殖、迁移及上皮间质转化产生抑制作用,同时可下调YAP1和Vimentin mRNA的表达,上调LATS1、MST1、E-钙黏蛋白mRNA的表达;该作用机制可能与其激活Hippo信号通路有关。

Objective To explore the effects of metformin on the proliferation,migration,and epithelial-mesenchymal transition(EMT)of human lens epithelial cells(LEC)induced by transforming growth factor-β_(2)(TGF-β_(2)).Methods Immortalized human LEC(HLEB-3 cells)was selected as the cell source.Human LEC with a cell fusion degree of 80%was cultured in DMEM low-glucose medium containing 10 mg·L^(-1)TGF-β_(2)for 24 hours as the control group.The cells treated with TGF-β_(2)and then further treated with different concentrations of metformin were used as the experimental group.After treatment,the morphological changes of cells in each group were observed under an inverted microscope.The cytotoxicity was detected by CCK-8 assay,and the cell survival rate was calculated.The expression levels of Yes-associated protein 1(YAP1),large tumor suppressor 1(LATS1),and Vimentin in cells were detected by Western blot.The mRNA expression levels of YAP1,LATS1,mammalian STE20-like kinase 1(MST1),Vimentin,and E-cadherin were detected by real-time quantitative polymerase chain reaction.Results The cytotoxicity test of metformin showed that when the concentration of metformin was greater than 15 mmol·L^(-1),the survival rate of human LEC significantly decreased,indicating that the concentration of metformin had a significant impact on the survival of LEC.Therefore,15 mmol·L^(-1)was selected for subsequent experiments.Metformin significantly inhibited the proliferation of human LEC induced by TGF-β_(2)in a dose-dependent manner(P<0.001).After 24 hours of treatment with 15 mmol·L^(-1)metformin,the relative expression levels of YAP1 and Vimentin proteins in human LEC were lower than those in the control group(both P<0.05),while the relative expression level of LATS1 protein was higher than that in the control group(P<0.05).After 24 hours of treatment with 15 mmol·L^(-1)metformin,the relative expression levels of YAP1 and Vimentin mRNA in human LEC were lower than those in the control group,while the relative expression levels of LATS1,MST1,and E-cadherin mRNA were higher than those in the control group,with statistically significant differences(all P<0.05).Conclusion Metformin can inhibit the proliferation,migration and EMT of human LEC induced by TGF-β_(2)in vitro,downregulate the expression of YAP1 and Vimentin mRNA,and upregulate the expression of LATS1,MST 1 and E-cadherin.The mechanism of action may be related to its activation of the Hippo signaling pathway.