The Molecular Mechanism Of Curcumin Inhibits Hepatocellular Carcinoma EMT Induced By TGFβ

Objective: This study was to investigate the effect of curcumin on TGFβ-induced epithelial-mesenchymal transition(EMT)in hepatocarcinoma cells and to study its molecular mechanism and provide effective targets and feasibility strategies for the prevention and treatment of hepatocellular carcinoma.Methods: 1)Establishment of EMT cell models: Two kinds of hepatoma cells HepG2 and QGY-7703 were used to establish the cell models of the TGFβ-induced EMT which providing the basic for subsequent experiments to observe cell morphology and detect EMT marker protein,such as E-cad,Vim and Fn by western blotting.2)Effect of TGFβ on invasion ability of hepatocellular carcinoma cells: Two kinds of hepatocellar carcinoma cell lines were cultured to detect the invasion ability by the scintigraphy experiment and transwell tumor cell migration experiment.3)To investigate the effect of curcumin on TGFβ-induced EMT in hepatocarcinoma cells: Each cell line was treated with or without curcumin and TGF β,then detected the cell morphology and the expression of EMT marker protein.4)To study the effect of curcumin on the invasion of TGFβ-induced EMT in hepatocellular carcinoma: Detect the invasion of hepatocellar carcinoma cells by the scintigraphy experiment and transwell tumor cell migration experiment.5)To clarifythe molecular mechanism of curcumin inhibiting EMT induced by TGFβin hepatocellular carcinoma: The expression of Snail and the phosphorylation of Smad2 were detected by Western blotting,and Smad2 nuclear translocation were detected by laser confocal.Detection the combination of smad2 and snail promoter by EMSA.The activation of snail promoter and its mRNA level were detected through snail promoter luciferase reporter gene assay and real-time quantitative PCR.Results: 1)Western blotting demonstrated that TGFβ upregulated the expression of Vim and Fn and downregulated the expression of E-cad in HepG2 and QGY-7703 cell lines,cells changed from epithelial to mesenchymal morphology,which indicated that EMT model was established successfully.2)Through the scratch test and transwell tumor cell migration experiments,we demonstrated that TGFβ can induce the invasion and metastasis of hepatocellular carcinoma cells.3)Western blotting and RT-PCR results showed that the expression of snail,Fn and Vim induced by TGFβ decreased significantly when pretreated with curcumin,indicating that curcumin can inhibit TGFβ-induced EMT in hepatocellular carcinoma.4)Through the scratches and transwell tumor cell migration experiments,it demonstrated that curcumin can inhibit TGFβ-induced invasion and metastasis.5)Laser confocal microscopy,luciferase reporter gene experiments and western blotting experiments demonstrated that the phosphorylation of smad2 and the mounts of smad2 enter into nucleus increased and the snail promoter was activated when treated with TGFβ alone,but when treated with curcumin and TGFβtogether,it can decrease all these event.Conclusions: 1)Establishment of the model of TGFβ-induced EMT is successful.2)Curcumin can inhibit EMT induced by TGFβ inhepatocarcinoma cells.3)Curcumin inhibited the TGFβ-induced EMT by inhibiting the phosphorylation of smad2 and nuclear translocation and preventing the smad2 combined with the promoter of snail,then downregulating the expression of snail to inhibit EMT induced by TGFβin hepatocarcinoma cells,which provided effective targets and feasibility strategies for the prevention and treatment of hepatocellular carcinoma.