The Role And Mechanism Of TGF-β And Ras Gene In The Process Of Epithelial–mesenchymal Transition In Hepatocarcinoma

OBJECTIVE: A lentiviral vector carrying Ha-ras gene and transfected MMH-D3 cell lines were constructed to observe the changes of cell moephology and expressions of cell adhesion molecule in MMH-D3 cell with Ras gene induced by TGF-β-1 and to investigate the role and mechanism of TGF-βand Ras genes in the Epithelial-Mesenchymal Transition of liver cells in mouse. We aimed to establish the model of hepatocarcinoma invasion and metastasis in vitro for the future research of the mechanism of hepatocarcinoma invasion and metastasis.METHODS: Lentiviral ventor with v-ha-ras gene was chemosynthesized, amplified, purified and extracted by chemical methods. The correct Ras gene was identified by endonucleases digestion and sequencing. Recombinant lentiviruses were produced by 293T cells followed by co-transfection of Ras gene plasmids and packaging plasmids. The supernatant of virus-producing cells containing Ras genes were used to transfect MMH-D3 cells. Labelling protein GFP in cells was measured by fluorescent microscopy. The GFP-positive cells were separated by flow cytometry, and the expreession of Ras was detected by western blot and RT-PCR in MMH-R cell lines (the cell lines containing the Ras gene). MMH-R cells were cultured by the culture medium containing TGF-β1.Culture medium was replaced the next day. Cell morphology was observed after 2-3 days and photographed. Cell morphological changes were observed and the expression of E-cadherin andβ-catenin were detected by immunofluorescence in MMH-D3 cells, MMH-R cells and MMH-R cells treated with TGF-β1 (MMH-RT cell lines). RESULTS: The recombinant plasmid was effectively transfected in 293T cells and green fluorescence could be observed under fluorescence microscopy in MMH-D3 cells from the supernatant of lentivirus effectively infected. Results of RT-PCR and western blot demonstrated that the expression and protein of Ras increased markedly in MMH-R cells compared to MMH-D3 cells. There was no significant morphological difference between MMH-R and MMH-D3 cells. But morphology of MMH-R cell, induced by TGF-β1, changed from epithelial phenotype to fiber phenotype which suggested Epithelial-Mesenchymal Transition. Results of immunofluorescence demonstrated that E-cadherin andβ-catenin expressed on the membrane of MMH-D3 and MMH-R cells, but in cytoplasm. The expression of E-cadherin andβ-catenin were decreased and scattered in cytomembrane and cytoplasm of transformed MMH-RT cell,CONCLUSION: TGF-β1 could induce epithelial mesenchymal - transition in hepatocytes of ras transformed mouse in vitro.