绿茶多酚通过miR-149/Smad2对TGF-β1诱导的人皮肤角质形成细胞增殖迁移的影响

Effects of green tea polyphenols on the proliferation and migration of human skin keratinocytes induced by TGF-β1 through miR-149/Smad2

目的:探讨绿茶多酚对转化生长因子β1(TGF-β1)诱导的人皮肤角质形成细胞增殖迁移的影响及其分子机制。方法:人皮肤角质形成细胞分为对照组、TGF-β1组、TGF-β1+绿茶多酚12.5μg·ml^(-1)组、TGF-β1+绿茶多酚25μg·ml^(-1)组、TGF-β1+绿茶多酚50μg·ml^(-1)组、TGF-β1+miR-149组、TGF-β1+miR-NC组、TGF-β1+绿茶多酚+anti-miR-149组;MTT法检测细胞活性;RT-qPCR检测miR-149表达水平;Western blot法检测Smad2蛋白表达;划痕实验检测细胞划痕愈合率;Transwell小室实验检测细胞迁移;双荧光素酶报告实验检测miR-149与Smad2的靶向关系。结果:TGF-β1诱导的人皮肤角质形成细胞中miR-149表达水平降低,Smad2表达水平升高,细胞活性降低,细胞划痕愈合率降低,迁移细胞数减少(P<0.05);不同浓度绿茶多酚处理后TGF-β1诱导的人皮肤角质形成细胞中miR-149表达水平升高,Smad2表达水平降低,细胞活性升高,细胞划痕愈合率升高,迁移细胞数增加(P<0.05)。过表达miR-149后,Smad2表达水平降低,细胞活性升高,细胞划痕愈合率升高,迁移细胞数增加(P<0.05)。抑制miR-149可逆转绿茶多酚对TGF-β1诱导的人皮肤角质形成细胞增殖迁移的影响。miR-149靶向调控Smad2。结论:绿茶多酚通过调控miR-149/Smad2促进TGF-β1诱导的人皮肤角质形成细胞增殖迁移。

Objective:To explore the effect of green tea polyphenols on the proliferation and migration of human skin keratinocytes induced by transforming growth factorβ1(TGF-β1)and the molecular mechanism.Methods:Human skin keratinocytes were divided into control group,TGF-β1 group,TGF-β1+green tea polyphenols 12.5μg·ml^(-1) group,TGF-β1+green tea polyphenols 25μg·ml^(-1) group,TGF-β1+green tea polyphenols 50μg·ml^(-1) group,TGF-β1+miR-149 group,TGF-β1+miR-NC group,TGF-β1+green tea polyphenol+anti-miR-149 group.MTT was used to detect cell viability;RT-qPCR was used to detect the expression level of miR-149;Western blot was used to detect the expression of Smad2 protein;scratch test was used to detect cell scratch healing rate;Transwell was used to detect cell migration;dual luciferase reporter was used to detect the targeting relationship between miR-149 and Smad2.Results:The expression of miR-149 in human skin keratinocytes induced by TGF-β1 was decreased,the expression of Smad2 was increased,the cell viability was decreased,the healing rate of cell scratches was decreased,and the number of migrating cells was decreased(P<0.05);after treated with different concentrations of green tea polyphenols,the expression level of miR-149 in human skin keratinocytes induced by TGF-β1 was increased,the expression level of Smad2 was decreased,the cell activity was increased,the healing rate of cell scratches was increased,and the number of migrating cells was increased(P<0.05).After overexpression of miR-149,Smad2 expression level was decreased,cell viability was increased,cell scratch healing rate was increased,and the number of migrating cells was increased(P<0.05).Inhibition of miR-149 reversed the effect of green tea polyphenols on the proliferation and migration of human skin keratinocytes induced by TGF-β1.miR-149 targets and regulates Smad2.Conclusion:Green tea polyphenols can promote the proliferation and migration of human skin keratinocytes induced by TGF-β1 by regulating miR-149/Smad2.