The effects of c-Src kinase on EMT signaling pathway in human lens epithelial cells associated with lens diseases

Background The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods Human lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1 alpha, IL-6, TNF-alpha or IL-1 beta, at 10 ng/mL or high glucose (35.5 mM) for 30 mins. Activity of c-Src kinase was evaluated by the expression of p-Src(418) with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-Src(Y530F) to upregulate activity of c-Src kinase, and pSlience4.1-ShSrc to knock it down. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, alpha-SMA, and Vimentin were examined at 48 h by RT-PCR and western blot. At 48 h and 72 h of transfection, cell proliferation was detected by MTT, and cell mobility and migration were determined by scratch and transwell assays. Results Activity of c-Src kinase, which causes the expression of p-Src(418), was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-Src(Y530F), the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src(418) increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and alpha-SMA were elevated on both mRNA and protein levels at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src(418) expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and alpha-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced. Conclusions The c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.