Smad4Influences SMMC-7721Cell Invasion And Metastasis By Regulating Epithelial-Mesenchymal Transition

Objective：The aim of this study is to investigate the influence of loss of Smad4expression onTGF-β1-induced epithelial-mesenchymal transition in the human hepatocellularcarcinoma cell line SMMC-7721and its molecular mechanism for the invasion andmetastasis of hepatocellular carcinoma cells. In order to provide a theoretical basis tofuther explain the hepatocellular carcinoma invasion and metastasis mechanism.Methods：①SMMC-7721cells were stimulated with different concentrations of TGF-β1, thenwe observed cell proliferation at different time points and counted the cells, drawingthe growth curve.②We observed the corresponding protein expression after knocking down the Smad4gene in the human hepatocellular carcinoma cell line SMMC-7721by Western blot；The influence of loss of Smad4expression on EMT-related markers E-cadherin,β-catenin and Vimentin total protein levels was evaluated by Western blot；③The corresponding changes of β-catenin and Vimentin mRNA expression weredetected by reverse-transcription PCR；④Immunofluorescence was used to analyze the location of Smad4，β-catenin，Vimentin and their fluorescence intensity in the human hepatocellular carcinoma cellline SMMC-7721and those transfection ones， named as RNAi-Smad4-2andRNAi-Smad4-12cells. The control groups were the human hepatocellular carcinoma cell line CON and the human hepatocellular carcinoma cell line RNAi-NC cells.Results：①Cell proliferation rate reached a peak after incubation with10ng/mLTGF-β1. Thenext experiments continued with this concentration and time point to treat the cells.②Compared with the human hepatocellular carcinoma cell line CON and RNAi-NCcells, the expression of Smad4protein expression and correspondingimmunofluorescence intensity remarkably decreased in the human hepatocellularcarcinoma cell line RNAi-Smad4-2and RNAi-Smad4-12.③The renal tubular epithelial cells were positive reference. E-cadherin protein wasnegative expression in the human hepatocellular carcinoma cell line CON, RNAi-NCcells and the transfection groups RNAi-Smad4-2.④Compared with the human hepatocellular carcinoma cell line CON and RNAi-NC(SMMC-7721cells infected with empty lentiviral vectors) cells，the expression ofβ-catenin mRNA and corresponding total protein expression remarkably increased inRNAi-Smad4-2and RNAi-Smad4-12(knocking down the Smad4expession)transfection groups(P<0.05); loss of Smad4expression promoted β-catenin nucleartranslocation. Immunofluorescence assay revealed that in the human hepatocellularcarcinoma cell line CON and RNAi-NC cells，β-catenin fluorescence expressionlocated in the nuclear；while in the RNAi-Smad4-2and RNAi-Smad4-12transfectiongroups, it located in the cytoplasm.⑤On the other hand，loss of Smad4expression down-regulated Vimentin proteinexpression(P<0.05)and cytoplasmic fluorescence intensity，but there is no significantdifference in its corresponding mRNA expression in the human hepatocellularcarcinoma cell line SMMC-7721cells and those transfection groups.Conclusion： Loss of Smad4expression plays an important role in inhibitingepithelial-mesenchymal transition through regulating EMT markers β-catenin andVimentin expression in the human hepatocellular carcinoma cell line SMMC-7721.Itmay affect the hepatocellular carcinoma invasion and metastasis.