DUSP6在TGF-β2诱导的晶状体上皮细胞间质转分化及细胞外基质合成中的作用

The role of DUSP6 in TGF-β2-induced epithelial-mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

目的探讨双特异性磷酸酶6(dual-specificity phosphatase 6,DUSP6)在转化生长因子β2(transforming growth factorβ2,TGF-β2)诱导的人晶状体上皮细胞(human lens epithelial cells,HLECs)上皮间质转分化(epithelial mesenchymal transition,EMT)及细胞外基质(extracellular matrix,ECM)合成的调控作用。方法用10μg/L TGF-β2处理晶状体上皮B3(HLE-B3)细胞株不同时间(0、12、24、48 h)后,实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹(Western blotting)方法检测DUSP6、EMT标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)以及ECM主要成分纤维连接蛋白(fibronectin,Fn)的表达。构建DUSP6过表达载体并转染HLE-B3细胞株后,加10μg/L TGF-β2处理24 h,分为3组:TGF-β2组、空载体(empty vector,EV)+TGF-β2组、DUSP6+TGF-β2组。RT-qPCR和Western blotting检测DUSP6过表达对α-SMA、Fn表达的影响,划痕实验和Transwell迁移实验用于评估DUSP6过表达对细胞迁移的作用。结果与对照组相比,10μg/L TGF-β2处理24 h时DUSP6 mRNA及蛋白表达均减少(P<0.05)。与EV+TGF-β2组相比,DUSP6+TGF-β2组抑制了α-SMA和Fn的表达与细胞迁移(P<0.05)。结论DUSP6能够抑制TGF-β2引起的晶状体上皮细胞EMT及ECM合成,靶向DUSP6可能为后发性白内障提供一种潜在的治疗策略。

Objective To investigate the role of dual-specific phosphatase 6(DUSP6)in the regulation of human lens epithelial cells(HLECs)epithelial-mesenchymal transition(EMT)and extracellular matrix(ECM)synthesis induced by transforming growth factorβ2(TGF-β2).Methods Human lens epithelial B3(HLE-B3)cells were treated with 10μg/L of TGF-β2 for 0 h,12 h,24 h and 48 h.Then we detected the expressions ofα-smooth muscle actin(α-SMA)and fibronectin(Fn)by real-time fluorescent quantitative PCR(RT-qPCR)and Western blotting.The overexpression vector of DUSP6 was constructed and transfected into HLE-B3 cell line and divided into three groups:TGF-β2,empty vector(EV)+TGF-β2,and DUSP6+TGF-β2.RT-qPCR and Western blotting were used to detect the effect of overexpression of DUSP6 on the expressions ofα-SMA and Fn.Scratch test and Transwell migration test were used to evaluate the effect of DUSP6 overexpression on cell migration.Results Compared with the control group,the expressions of DUSP6 mRNA and protein decreased after 10μg/L of TGF-β2 treatment for 24 h(P<0.05).Compared with EV+TGF-β2 group,DUSP6+TGF-β2 group inhibited the migration of HLE-B3 cells and the expressions ofα-SMA and Fn(P<0.05).Conclusion DUSP6 could inhibit EMT and ECM synthesis in lens epithelial cells induced by TGF-β2,which might provide a potential therapeutic strategy for posterior capsule opacification.