Effect Of TGF-β1 On Epithelial-mesenchymal Transition And Invision Ability Of AGS Cells

ObjectiveThis experiment was designed to investigate the effect of tranforming growth factor beta-1 (TGF-β1) on epithelial-mesenchymal transition(EMT) and invasion ability of human gastric adenocarcinoma cell line AGS in vitro.Methods1.AGS cells cultured under normal conditions in vitro were treated with 10ng/ml TGF-β1 interference,after three-generation continuous culture,morphological changes of AGS cells were observed under inverted microscope.2.Set up the blank control group, the experimental group were added to 1,2,5,10,20,50 ng/ml final concentration of TGF-β1 for 24 hours of continuous culture. To analyse the proliferation of AGS cells by MTT colorimetric assay.3.After the AGS cells were fully integrated, scratched vertically in the single-cell with 200μl micro-pipette. In the experimental group was added to a final concentration of 1 Ong/ml of TGF-β1.Scratch healing was observed under inverted microscope every 24 hours compared with the control group.4.Applicated with 24-well Transwell invasion chambers for AGS cell invasiveness test. And the experimental group was added to a final concentration of 10ng/ml of TGF-β1,the control group was added to equal amount of PBS as a chemotactic agent. Removed Transwell invasion chambers after cultured 24 hours continuously,fixed with 4% paraformal dehyde, after hematoxylin staining,counted cells on the back of transmembrane membrane with high-powered microscope. Compared with the control group to detect cell motility and invasiveness of the change.5.Seeded AGS cells with exponential growth. Added a final concentration of lOng/ml of TGF-β1 in the experimental group. After 48 hours,detected expression of nuclear transcription factor Snail, epithelial cell marker protein E-cadherin and mesenchymal cell marker protein N-cadherin with immunofluorescence method.6.Cultured the AGS cells with a final concentration of 1Ong/ml of TGF-β1 for 48 hours. Detected the expression changes of Snail, E-cadherin and N-cadherin in the experimental group and blank control group with Western blot.Results1.TGF-β1 induced AGS cells morphological alteration from epithelial to mesenchymal. The AGS cells sheded their epithelial multilateral traits,and shifted to fibroblastic morphology:some cells showed fusiform shape, and the arrangement of the cells showed disorderly.2.MTT assay results:1,2,5,10 ng/ml concentrations of TGF-β1 could promote cell proliferation, and cell proliferation and TGF-β1 concentration showed the linear relationship. As the concentration increased,cell proliferation was more obviously. When the concerntration of TGF-β1 was 1Ong/ml, the role was the strongest. But the at the concentrations 20,50 ng/ml of TGF-β1 the ratio of AGS proliferation was decreased gradually.3.Cell scratch test results:Compared with the control group,cell migration ability was enhanced significantly in the experimental group added to 1Ong/ml TGF-β1 to culture continuously.4.Transwell invasion assay results:Cultured cells in Transwell chamber after 24 hours, trans-membrane cell number of the AGS cell was 58.33±5.859. And in the experimental group, trans-membrane cell number was 105.67±4.041, differences between the two was significant (p<0.05). It showed that the motility and invasive ability of AGS cells was greatly enhanced.5.Immunofluorescence detection the expression of Snail, E-cdaherin and N-cadherin:Added to AGS cells with rabbit anti-human Snail (1:800) by adding specific goat anti-rabbit IgG (H+L)/FITC secondary antibody. We found that AGS cell nucleus showed green fluorescence with the fluorescence microscope observation. The experimental group showed a stronger expression of green fluorescence compared with the control group. Added to AGS cells with mouse anti-N-cadherin monoclonal antibody by adding goat anti-mouse IgG (H+L)/TRITC labeled fluorescent secondary antibody, we found that the AGS cells showed red fluorescence. The experimental group showed a stronger expression of red fluorescence compared with the control group. Added to AGS cells with mouse anti-E-cadherin monoclonal antibody by adding goat anti-mouse IgG (H+L)/TRITC labeled fluorescent secondary antibody, we did not found that the AGS cells show red fluorescence. The experimental group did not detect the expression of red fluorescent compared with the control group too.6. Western blot results:Compared with the control group, the expressions of Snail and N-cadherin were increased in the experimental group added to lOng/ml TGF-β1. We also detected that the expression of E-cdaherin in the AGS cells was weak,and by adding TGF-β1, E-cadherin expression was decreased and weaker.ConclusionOur study showed that TGF-β1induced AGS cells with epithelial to mesenchymal transition, and its motility and invasion ability were enhanced.