Development Of A Dual-color Fluorescence Cell Model To Visualize TGF-β Induced EMT

Epithelial-mesenchymal transition (EMT), which down-regulates epithelial traits and acquires mesenchymal traits, plays critical roles in development, wound healing, and stem cell behavior, and pathologically to fibrosis and cancer progression through a variety of mechanism. Among signaling pathways, transforming growth factor-β (TGF-β) family signaling has a predominant role. Although some efficient systems for EMT have been established, robust functional tools to monitor TGF-β induced EMT remains lacking.Due to the important role of EMT in the development and pathological conditions such as cancer progression, fibrosis, and chronic inflammation, it is very important to establish a rapid screening model for EMT. Therefore we reported a rapid visual cell model by using of EMT-associated markers which can successfully show the process of EMT. In fact, the down-regulation of E-cadherin and the up-regulation of Vimentin is the most dominant feature of EMT. We constructed E-cadherin promoter(-1000---+125bp) driven GFP and Vimentin promoter (-1027---+1121bp) driven RFP plasmids. After the transfection of human epithelial cell MCF10A with these two reporter plasmids separately, we got two kinds of stable cell lines, E-cadherin promotor driven GFP cell line and Vimentin promotor driven RFP cell line. Then we picked up two E-cad-GFP clones (M6and13G) and two Vim-RFP clones (ZB1and ZB101), and all of them showed the good respond of TGF-P mediated EMT. In order to better monitor the process of EMT, we combined these four clones and finally choose the ideal combination----M6+ZB1and13G+ZB101. These two combination all showed green florescence when the cell had an epithelial phenotypes. After TGF-β48hours treatment, cells of those two combinations lose epithelial cell-specific phenotypes and develop features of mesenchymal cells, and as a result the cells turn from green to red fluorescence.. And western blot results also demonstrated the desired functions of our dual reporter system. Except TGF-β treatment,these two combinations also can show good response to TNF-a, VEGF, EGF and PDGF-BB. With these treatments, western blot showed the down-regulation of E-cadherin and the up-regulation of Vimentin, N-cadherin, at the same time the cells turn to the red fluorescence. So we determined that these two cell combination model can be used to quickly facilitate detection of EMT process.In summary, our experiments offers an easy-to-use tool for monitoring EMT in mammalian cells by changing the color of fluorescence. It can be used in rapid screening of the genes and small molecule which affect the process of EMT. This study provides a better prospect for drug screening.