The Role and Mechanism of TGF-β and Ras Gene in the Process of Epithelial–mesenchymal Transition in Hepatocarcinoma
|School||Huazhong University of Science and Technology|
|Keywords||Epithelial cells - mesenchymal transition Ras gene Transforming growth factor β1 MMH-D3 cells|
Objective: In this study, by constructing the lentiviral vector carrying the Ha-ras gene and transfected into mouse liver pluripotent stem cells were observed in the transforming growth factor (TGFβ-1) induced pluripotent stem cells by the Ha-ras transformed mouse liver cell morphology and surface adhesion molecule expression change, and to explore the role and possible mechanisms of TGFβ-1 and Ras gene in liver cells epithelial cells - mesenchymal cell transformation (Epithelial-Mesenchymal Transition, EMT), through the means of building invasion and metastasis of hepatocellular carcinoma cells in vitro model, an experimental basis for further study of the liver cells in tumor invasion and metastasis. Method: chemical synthesis plasmid containing v-ha-ras gene amplified purified extraction and identification, to carry the target gene plasmid and packaging plasmids were co-transfected 293T cells, recombinant lentivirus. Cultured in vitro MMH-D3 mouse liver cells, to take viral supernatant infection MMH-D3 cells, fluorescence microscopy green fluorescent protein expression in the target cells; isolated GFP-positive cells by flow cytometry subculture, using RT-PCR ras gene in MMH-R cells (MMH-D3 cell line) expression of ras gene expression and Western blot. MMH-R cells with culture medium containing TGF-β1, the next day, the medium was replaced, and cultured for 2-3 days after cell morphology was observed and photographed, immunofluorescence staining of E-cadherin and β-catenin in MMH-D3 cells, MMH- The R cells MMH-RT cells (TGF-β1 intervention MMH-R cells to form cell line) in the expression. Results: The recombinant lentivirus high efficiency transfection of 293T cells MMH-D3 cells transfected charged lentiviral supernatant, visible under a fluorescence microscope a lot of green fluorescence. RT-PCR and Western blot results showed that MMH-R cells ras gene and protein expression was significantly increased, a significant difference (P lt; 0.05) compared with the control group MMH-D3 cell lines. Optical microscope show no significant difference in MMH-R cells transfected by lentivirus normal MMH-D3 cell morphology, cell morphology MMH-RT TGF-β1 intervention change significantly, spindle cells transformed by epithelioid phenotype shaped into a fiber-like phenotype cells, epithelial cell mesenchymal cell transformation (EMT) occurs. Immunofluorescence staining cells MMH-D3 and MMH-R cells, E-cadherin and β-catenin in the cell membrane expressed abundantly expressed in the cytoplasm less; MMH-RT cells transformed occurred E-cadherin and β-catenin expression were significantly reduced cytoplasmic membrane were scattered. Conclusion: TGF-β in vitro induced ras transformed mouse liver cells epithelial cells - mesenchymal transition.