MiR-125b对心肌梗死后成纤维细胞的调控机制研究

Regulation mechanism of miR-125b to cardiac fibroblasts after myocardial infarction

目的探讨微小核糖核酸125b(miR-125b)对成人心脏成纤维细胞(HCF-a)合成胶原、增殖、活化、迁移、凋亡的调控作用及对转化生长因子β1(TGF-β1)通路的影响。方法体外培养HCF-a细胞,分为空白对照组和miR-125b模拟物转染组。采用RT-PCR和Western blot法检测胶原-Ⅰ、胶原Ⅲ、α-平滑肌肌动蛋白(α-SMA)、TGF-β1、p-Smad2、p-Smad3基因和蛋白表达情况;采用MTT法检测HCF-a细胞增殖情况;采用Transwell细胞迁移实验检测细胞迁移能力;采用流式细胞术检测细胞凋亡情况。结果与空白对照组相比,miR-125b过表达可明显上调Col-Ⅰ和Col-Ⅲ、α-SMA m RNA和蛋白的表达,促进细胞增殖、迁移和凋亡(P<0.05)。另外,经RT-PCR检测,与空白对照组比较,miR-125b过表达可明显促进TGF-β1 m RNA的表达水平(P<0.05);经Western blot检测,与空白对照组比较,miR-125b过表达对HCF-a细胞Smad2/Smad3蛋白磷酸化水平均有明显上调作用(P<0.05)。结论Mi R-125b可以通过调控TGF-β1/Smad通路参与心肌纤维化进程。

Objective To investigate the regulation effect of microRNA 125b(miR-125b)on synthesis,proliferation,activation,migration and apoptosis of human cardiac fibroblast-adult HCF-a(HCF-a),and influence of miR-125b on transforming growth factorβ1(TGF-β1)pathway.Methods HCF-a was cultured in vitro,and then divided into blank control group and miR-125b group.The levels of collagen-I(Col-I),collagen-III(Col-III),α-smooth muscle actin(α-SMA)and TGF-β1,and mRNA and protein expressions of phosphorylated Smad2(p-Smad2)and phosphorylated Smad3(p-Smad3)were detected by using real-time fluorescence quantitative polymerase chain reaction(RT-PCR)and Western blotting assay.The proliferation of HCF-a was detected by using methyl thiazolyl tetrazolium test(MTT),migration ability of HCF-a was detected by using Transwell cell migration test,and apoptosis of HCF-a was detected by using flow cytometry(FCM).Results The mRNA and protein expressions of Col-I,Col-III andα-SMA were significantly up-regulated,and proliferation,migration and apoptosis of HCF-a were improved in miR-125b group compared with blank control group(P<0.05).The results of RT-PCR showed that mRNA expression of TGF-β1 was significantly improved in miR-125b group compared with blank control group(P<0.05).The results of Western blotting assay showed that level of p-Smad2/p-Smad3 was significantly up-regulated in test group compared with blank control group(P<0.05).Conclusion MiR-125b can take part in myocardial fibrosis process through regulating TGF-β1/Smad pathway.