microRNA-378b对心脏成纤维细胞纤维化水平的影响及机制研究

Effect of microRNA-378b on cardiac fibroblasts fibrosis and its mechanism

目的研究microRNA-378b(miR-378b)对心脏成纤维细胞的纤维化水平的影响及分子机制。方法小鼠行慢性心肌梗死手术构建体内心肌纤维化模型,利用转化生长因子-β(TGF-β)诱导原代心脏成纤维细胞发生纤维化以构建体外心肌纤维化模型;实时荧光定量PCR(qRT-PCR)法检测2种纤维化模型中miR-378b的表达水平;Western blotting法检测miR-378b模拟物和抑制物对心脏成纤维细胞α-平滑肌肌动蛋白(α-SMA,心肌纤维化特异性指标)表达量的影响;TargetScan、miRDB和miRWalk软件预测miR-378b的下游靶基因,双荧光素酶实验验证miR-378b与生长相关蛋白43(GAP43)的靶向关系;Western blotting法检测miR-378b模拟物和抑制物对心脏成纤维细胞GAP43表达的影响;Western blotting法检测体内和体外2种纤维化模型中GAP43的蛋白水平。结果小鼠心肌纤维化模型组miR-378b表达量较假手术组显著降低(P<0.01),心脏成纤维细胞TGF-β处理组miR-378b表达量较对照组显著降低(P<0.001)。在基础水平和TGF-β处理后,与对照模拟物组比较,miR-378b模拟物显著降低细胞的α-SMA蛋白水平(P<0.05);在基础水平,与对照抑制物组比较,miR-378b抑制物可显著升高细胞的α-SMA蛋白水平(P<0.05);但在TGF-β处理的细胞中,miR-378b抑制物不能进一步增加α-SMA蛋白表达量。GAP43是miR-378b直接作用的下游靶基因,与对照组比较,miR-378b可负调控心脏成纤维细胞GAP43的蛋白表达水平(P<0.01)。心肌纤维化模型组小鼠心肌GAP43蛋白表达量较假手术组显著升高(P<0.01),心脏成纤维细胞TGF-β处理组GAP43蛋白表达量较对照组显著升高(P<0.001)。结论miR-378b可抑制心脏成纤维细胞纤维化,该功能可能通过抑制其下游靶基因GAP43发挥作用。

Objective To investigate the effect of microRNA-378b(miR-378b)on the fibrosis level of cardiac fibroblasts and its molecular mechanism.Methods The model of myocardial fibrosis in vivo was established by operation of chronic myocardial infarction in mice,transforming growth factor-β(TGF-β)was used to treat cardiac fibroblasts to construct myocardial fibrosis model in vitro.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression levels of miR-378b in two fibrosis models.The effects of miR-378b mimics and inhibitors onα-smooth muscle actin(α-SMA,a specific indicator of myocardial fibrosis)expression of cardiac fibroblasts were detected by Western blotting.The downstream target genes of miR-378b were predicted by three softwares(TargetScan,miRDB,and miRWalk),double luciferase experiment verified the targeting relationship between miR-378b and GAP43.Western blotting was used to detect the effects of miR-378b mimics and inhibitors on GAP43 in cardiac fibroblasts.The protein levels of GAP43 in two fibrosis models were detected by Western blotting.Results The expression of miR-378b in the mouse myocardial fibrosis model group was significantly decreased compared with the sham operation group(P<0.01),and the expression of miR-378b in the cardiac fibroblast TGF-βtreatment group was significantly decreased compared with the control group(P<0.001).After treatment with TGF-βand in basal level,miR-378b mimics significantly decreasedα-SMA protein level compared with control mimics group(P<0.05).At the basal level,compared with the control group,miR-378b inhibitors significantly increased theα-SMA protein level of cells(P<0.05).However,miR-378b inhibitors did not further increaseα-SMA protein expression in TGF-β-treated cells.GAP43 was a downstream target gene directly affected by miR-378b.Compared with the control group,miR-378b can negatively regulate the protein expression level of GAP43 in cardiac fibroblasts(P<0.01).Compared with sham operation group,the expression level of GAP43 protein in myocardial fibrosis model group was significantly increased(P<0.01),and that in cardiac fibroblast TGF-βtreatment group was significantly increased compared with control group(P<0.001).Conclusion miR-378b can inhibit fibrosis level of cardiac fibroblasts,which may play a role through its downstream target gene GAP43.