miR-34a-5p对ARPE-19细胞TGF-β/Smad通路相关蛋白表达及细胞增殖、迁移和上皮-间质转化的影响

Effects of miR-34a-5p on transforming growth factor-β/Smad pathway-related protein expression, proliferation, migration and epithelial-mesenchymal transition of ARPE-19 cells

目的探讨miR-34a-5p对ARPE-19细胞TGF-β/Smad通路相关蛋白表达及细胞增殖、迁移和上皮-间质转化(EMT)的影响。方法将ARPE-19细胞分为4组:对照组、转化生长因子(TGF)-β1组、miR-34a-5p过表达组与复合干预组,对照组与TGF-β1组采用Lipofectamine 2000转染试剂转染对照模拟物,miR-34a-5p过表达组与复合干预组转染miR-34a-5p模拟物,培养24 h后采用无血清培养基饥饿培养12 h,TGF-β1组与miR-34a-5p过表达组均添加10 mg·L^(-1)的TGF-β1,复合干预组添加10 mg·L^(-1)的TGF-β1与10μmol·L^(-1)的TGF-β通路激活剂SRI-011381,各组细胞继续培养24 h,测定细胞增殖、迁移与EMT。采用Western blot检测细胞TGF-β/Smad通路相关蛋白的表达水平,Ki67荧光染色检测细胞增殖情况,免疫荧光染色测定细胞EMT相关蛋白表达情况,划痕实验和Transwell实验测定细胞的迁移和侵袭能力。结果与对照组相比,TGF-β1组细胞内TGF-β受体(TGF-βR)1、TGF-βR2、磷酸化Smad2(p-Smad2)和p-Smad3的表达水平均增高(均为P<0.05)。与TGF-β1组相比,过表达miR-34a-5p能够明显逆转TGF-β1对TGF-β/Smad信号通路的激活作用。与miR-34a-5p过表达组相比,复合干预组细胞内TGF-β/Smad信号通路明显被激活。与TGF-β1组相比,miR-34a-5p过表达组细胞Ki67阳性率为(6.67±1.52)%,明显降低(P<0.05)。与miR-34a-5p过表达组相比,复合干预组细胞Ki67阳性率为(16.67±1.53)%,显著升高(P<0.05)。划痕实验和Transwell实验结果显示:与对照组相比,TGF-β1能够促进ARPE-19细胞迁移和侵袭。过表达miR-34a-5p能够明显抑制TGF-β1组ARPE-19细胞的迁移和侵袭能力。在复合干预组内,TGF-β通路激活剂SRI-011381能够明显逆转过表达miR-34a-5p对ARPE-19细胞迁移能力的抑制作用。免疫荧光染色结果显示:与对照组相比,TGF-β1能够明显提高ARPE-19细胞内α-SMA和纤维连接蛋白表达水平,并抑制钙黏蛋白E的表达。与TGF-β1组相比,过表达miR-34a-5p能够明显抑制ARPE-19细胞发生EMT。在复合干预组内,TGF-β通路激活剂SRI-011381能够缓解过表达miR-34a-5p对ARPE-19细胞EMT的抑制作用。结论miR-34a-5p通过靶向失活TGF-β/Smad信号通路抑制ARPE-19细胞的增殖、迁移与EMT。

Objective To investigate the effects of miR-34a-5p on transforming growth factor(TGF)-β/Smad pathway-related protein expression,proliferation,migration,and epithelial-mesenchymal transition(EMT)of ARPE-19 cells.Methods ARPE-19 cells were divided into the control group,TGF-β1 group,miR-34a-5p overexpression group,and compound intervention group.Cells in the control group and the TGF-β1 group were transfected with Lipofectamine 2000 transfection reagent,and cells in the miR-34a-5p overexpression group and the compound intervention group were transfected with miR-34a-5p mimics.All these cells were starved in serum-free medium for 12 h after being cultured for 24 h.Cells in the TGF-β1 group and the miR-34a-5p overexpression group were intervened with TGF-β1(10 mg·L^(-1)),and cells in the compound intervention group were treated with TGF-β1(10 mg·L^(-1))and TGF-βpathway activator SRI-011381(10μmol·L^(-1)).All cells in the four groups were further cultured for 24 h.Cell proliferation,migration and EMT were measured.The TGF-β/Smad pathway-related protein level was detected by Western blot,and the EMT-related protein level was measured by immunofluorescence staining.Cell proliferation was assessed by Ki67 fluorescence staining,and cell migration and invasion were evaluated by scratch assay and Transwell assay.Results Compared with the control group,the ex-pression levels of TGF-βreceptor(TGF-βR)1,TGF-βR2,phosphorylated Smad2(p-Smad2)and p-Smad3 in the TGF-β1 group all increased(all P<0.05).Compared with the TGF-β1 group,overexpression of miR-34a-5p could significantly reverse the activation of the TGF-β/Smad signaling pathway by TGF-β1.Compared with the miR-34a-5p overexpression group,the TGF-β/Smad signaling pathway was significantly activated in the compound intervention group.The Ki67-positive rate in the miR-34a-5p overexpression group was(6.67±1.52)%,which was significantly lower than the TGF-β1 group(P<0.05).The Ki67-positive rate in the compound intervention group was(16.67±1.53)%,which was significantly higher than the miR-34a-5p overexpression group(P<0.05).The scratch and Transwell assay results showed that compared with the control group,TGF-β1 could promote migration and invasion of ARPE-19 cells.Overexpression of miR-34a-5p could significantly inhibit the migration and invasion of ARPE-19 cells in the TGF-β1 group.In the compound intervention group,the TGF-βpathway activator SRI-011381 could significantly reverse the inhibitory effect of overexpression of miR-34a-5p on ARPE-19 cell migration.The immunofluorescence staining results showed that compared with the control group,TGF-β1 could significantly increase the expression levels ofα-SMA and fibronectin in ARPE-19 cells and inhibit the expression of cadherin E.Compared with the TGF-β1 group,overexpression of miR-34a-5p could significantly inhibit the occurrence of EMT in ARPE-19 cells.In the compound intervention group,the TGF-βpathway activator SRI-011381 could alleviate the inhibitory effect of the overexpression of miR-34a-5p on EMT in ARPE-19 cells.Conclusion miR-34a-5p inhibits proliferation,migration and EMT of ARPE-19 cells by inactivating the TGF-β/Smad signaling pathway.