低表达Smad4对肝癌细胞SMMC-7721上皮间质转化的影响

目的:探讨低表达Smad4调控上皮间质转化影响肝癌细胞(hepatocellular carcinoma cells,HCC)侵袭转移的相关机制.方法:采用Western blot法观察低表达Smad4对β-catenin和Vimentin总蛋白表达水平的影响;逆转录PCR法测定低表达Smad4后,β-catenin和VimentinmRNA表达的变化;细胞免疫荧光法检测Smad4、β-catenin和Vimentin在肝癌细胞SMMC-7721及其转染组中的定位及荧光表达强度.结果:低表达Smad4后,相较于CON组和RNAi-NC组,在转染组RNAi-Smad4-2和RNAi-Smad4-12细胞中,β-cateninmRNA和相应总蛋白的表达水平明显增加(P<0.05),并促使其发生了核转位的改变;另一方面,在RNAi-Smad4-2和RNAi-Smad4-12两转染组细胞中,Vimentin的蛋白表达水平和相应胞浆荧光的表达强度比对照组明显下调(P<0.05),相应mRNA的表达水平在肝癌SMMC-7721细胞及其转染组无显著差异.结论:低表达Smad4可调控上皮间质转化相关标志物β-catenin和Vimentin的表达,发挥抑制肝癌SMMC-7721细胞上皮间质转化的作用.

Loss of Smad4 expression inhibits epithelialmesenchymal transition in SMMC-7721 cells

AIM:To investigate the influence of loss of Smad4 expression on TGF-β1-induced epithelialmesenchymal transition in the human hepatocellular carcinoma cell line SMMC-7721.METHODS:The influence of loss of Smad4 expression on the expression of β-catenin and Vimentin mRNAs and proteins was evaluated by RT-PCR and Western blot.Immuno fluorescence was used to analyze the location and fluorescence intensity of Smad4,β-catenin,Vimentin in non-transfected SMMC-7721 cells and those transfected with Smad-specific siRNAs(RNAiSmad4-2 and RNAi-Smad4-12) or unspecific siRNA(RNAi-NC).RESULTS:Compared to non-transfected SMMC-7721 cells and those tranfected with RNAi-NC,the expression of β-catenin mRNA and protein remarkably increased in SMMC-7721 cells transfected with RNAi-Smad4-2 or RNAiSmad4-12(all P < 0.05).Loss of Smad4 expression promoted β-catenin nuclear translocation.Immunofluorescence assay revealed that β-catenin fluorescence was located in the nuclei of nontransfected SMMC-7721 cells and those tranfected with RNAi-NC,but in the cytoplasm of SMMC-7721 cells transfected with RNAi-Smad4-2 or RNAi-Smad4-12.On the other hand,loss of Smad4 expression down-regulated Vimentin protein expression(P < 0.05) and cytoplasmic fluorescence intensity,but had no signif icant impact on Vimentin mRNA expression in SMMC-7721 cells and those transfected with different siRNAs.CONCLUSION:Loss of Smad4 expression regulates β-catenin and Vimentin and therefore plays an important role in inhibiting epithelial-mesenchymal transition in SMMC-7721 cells.