Abstract
BackgroundThe 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.MethodsHuman lens epithelial cells, HLE-B3, were exposed to either an inflammatory factor, specifically IL-1α, IL-6, TNF-α or IL-1β, 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-Src418 with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F 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, α-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.ResultsActivity of c-Src kinase, which causes the expression of p-Src418, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src418 increased. The expressions of both E-cadherin and ZO-1 were suppressed, while the expressions of vimentin and α-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-Src418 expressions were knocked down. The expressions of E-cadherin and ZO-1 increased, but the expressions of Vimentin and α-SMA decreased; meanwhile, cell proliferation, mobility and migration reduced.ConclusionsThe c-Src kinase in lens epithelial cells is easily activated by external stimuli, resulting in the induction of cell proliferation, mobility, migration and EMT.
Highlights
The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells
Inflammatory factors and high glucose activated c-Src kinase Using western blot assay, we found that after treatment with inflammatory factors IL-1α, IL-6, TNF-α and IL-1β for 30 mins, the activity of c-Src kinase in HLE-B3 cells was enhanced significantly compared with the control group
The expression of p-Src418 in the 35.5 mM glucose group was significantly higher than that in the 5.5 mM glucose group and mannitol group, whereas that in the mannitol group was almost the same as in the 5.5 mM glucose group (Fig. 1b). These results suggested that both inflammatory factors and high glucose stimulated the activity of cSrc kinase in HLE-B3 cells
Summary
The signaling pathway of epithelial to mesenchymal transition (EMT) is regulated by c-Src kinase in many cells. Li et al BMC Ophthalmology (2019) 19:219 diabetes at 6 and 12 months after cataract extraction [9] These studies suggest that inflammatory factors and high glucose are the stimulating factors for fibrosis of LECs. EMT is associated with many molecular and morphologic changes to epithelial cells that enable them to lose their cell polarity and cell-cell adhesion, gain properties in migration and invasion and become mesenchymal cells [10, 11]. The most marked characteristics of EMT are loss of epithelial markers, such as E-cadherin and ZO-1, and acquisition of a spindle shape cell, which is accompanied by accumulation of Vimentin and asmooth muscle actin (a-SMA) [12] This specific process is present in embryonic development, wound healing and tissue repairment and tumor metastasis. In organ fibrosis such as renal fibrosis, pulmonary fibrosis, hepatic fibrosis and ocular fibrosis, EMT is triggered by various biomolecules and signaling pathways, such as transforming growth factor-β (TGF-β) [13], insulin-like growth factor-1 (IGF-1) [14], transcription factor snail [15], and PI3K/Akt/mTOR/NF-κB signaling [16]
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