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 on α -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-β treatment group was significantly decreased compared with the control group (P<0.001). After treatment with TGF-β and in basal level, miR-378b mimics significantly decreased α-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 the α-SMA protein level of cells (P<0.05). However, miR-378b inhibitors did not further increase α-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-β treatment 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.

R542.2

南京市卫生科技发展专项资金项目计划(YKK19052)