Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, Shaanxi, China.
BMC Ophthalmol. 2019 Nov 8;19(1):219. doi: 10.1186/s12886-019-1229-4.
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.
Human 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-Src with western blot assay. To investigate the effects of activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-Src 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.
Activity of c-Src kinase, which causes the expression of p-Src, was upregulated by different inflammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-Src, the expression of c-Src kinase was upregulated on both mRNA and protein levels, and activity of c-Src kinase, expression of p-Src 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-Src 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.
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.
上皮间质转化（EMT）的信号通路在许多细胞中受 c-Src 激酶调控。本研究旨在探讨 c-Src 激酶在不同因素刺激下人眼晶状体上皮细胞 EMT 中的作用。
用炎性因子（10ng/ml 的 IL-1α、IL-6、TNF-α 或 IL-1β）或高糖（35.5mM）处理人晶状体上皮细胞系 HLE-B3 30min，以激活 c-Src 激酶。用 Western blot 检测磷酸化 Src（p-Src）的表达来评估 c-Src 激酶的活性。为了研究 c-Src 激酶激活对 EMT 的影响，用 pCDNA3.1-Src 转染 HLE-B3 细胞以上调 c-Src 激酶的活性，用 pSlience4.1-ShSrc 转染以敲低 c-Src 激酶的活性。转染 48h 后，通过 RT-PCR 和 Western blot 检测 c-Src 激酶和 EMT 分子标志物（E-cadherin、ZO-1、α-SMA 和 Vimentin）的表达。转染 48h 和 72h 后，通过 MTT 检测细胞增殖，通过划痕和 Transwell 检测细胞迁移和迁移能力。
不同炎性因子和高糖均可激活 HLE-B3 细胞中的 c-Src 激酶，导致 p-Src 的表达增加。当 HLE-B3 细胞用 pCDNA3.1-Src 转染时，c-Src 激酶的表达在 mRNA 和蛋白水平均上调，c-Src 激酶的活性和 p-Src 的表达增加。E-cadherin 和 ZO-1 的表达同时受到抑制，而 vimentin 和 α-SMA 的表达在 mRNA 和蛋白水平均上调。c-Src 激酶的激活伴随着细胞增殖、迁移和迁移能力的增加。相反，当 HLE-B3 细胞用 pSlience4.1-ShSrc 转染时，c-Src 激酶和 p-Src 的表达均被敲低。E-cadherin 和 ZO-1 的表达增加，而 vimentin 和 α-SMA 的表达减少；同时，细胞增殖、迁移和迁移能力降低。
晶状体上皮细胞中的 c-Src 激酶很容易被外部刺激激活，导致细胞增殖、迁移、迁移和 EMT 的诱导。
