miR-223对大鼠心肌细胞中纤维化相关信号通路分子的干预作用及机制

Effect and mechanism of miR-223 on fibrosis-related signaling pathway in rat cardiomyocytes

目的探讨miR-223对大鼠心肌细胞纤维化相关通路的保护作用及对Twist家族碱性螺旋-环-螺旋转录因子1(Twist1)、转化生长因子β1受体2(TGFBR2)的表达调控。方法体外培养大鼠心肌细胞H9c2,以TGF-β人工诱导心肌细胞纤维化相关通路活化,分为模型组、miR-223组(转染miR-223过表达慢病毒)以及miR-223-NC组(转染miR-223-NC慢病毒),同时设立正常细胞对照组。免疫组化检测α-SMA的表达情况;实时荧光定量PCR(real-time PCR)测定心肌细胞miR-223、collagenⅠ、collagenⅢ、Twist1、TGFBR2 mRNA表达水平;Western印迹法检测心肌细胞Twist1、TGFBR2、collagenⅠ、collagenⅢ及α-SMA蛋白水平;双荧光素酶报告基因验证miR-223对Twist1、TGFBR23′UTR的靶向调控。结果miR-223组α-SMA阳性心肌细胞平均吸光度(0.089±0.013)明显低于模型组和miR-223-NC组(0.134±0.018,0.132±0.016);miR-223组心肌collagenⅠ、collagenⅢ、Twist1、TGFBR2 mRNA表达水平与模型组及miR-223-NC组比较显著下降,且差异具有统计学意义(P<0.05);Twist1、TGFBR2、collagenⅠ、collagenⅢ及α-SMA蛋白水平较模型组及miR-223-NC组显著下降,且差异具有统计学意义(P<0.05);Twist1、TGFBR23′UTR野生型双荧光素酶报告质粒与miR-223 mimics共转染293T细胞,荧光素酶活性均显著降低[(0.48±0.06,0.51±0.07)比(0.92±0.17,0.94±0.12)]。结论miR-223可能通过下调Twist1、TGFBR2基因的表达抑制心肌细胞中纤维化相关信号通路活化。

Objective To investigate the protective effect of microRNA 223 (miR-223) on cardiac fibrosis-related signaling pathway and its regulation on expression of Twist family basic helix-loop-helix transcription factor 1 (Twist1) and transforming growth factor-β1 receptor 2 (TGFBR2) in rat cardiomyocytes.Methods Rat cardiomyocytes (H9c2) were cultured in vitro and treated with TGF-β to induce myocardial fibrosis. The miR-223 group was transfected with miR-223 lentivirus and miR-223-NC group was transfected with miR-223-NC lentivirus. Model group and blank control group had no transfection. Immunocytochemistry staining of alpha-smooth muscle actin (α-SMA) was used to calculate myocardial fibrosis. The mRNA level of miR-223, collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 were detected by real-time PCR. The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA were detected by Western blot. Target regulation of miR-223 on Twist1 and TGFBR2 3′UTR was verified by double luciferase reporter gene system.Results The average optical density of α-SMA-positive cardiomyocytes in miR-223 group (0.089±0.013) was significantly lower than that in model group and miR-223-NC group (0.134±0.018, 0.132±0.016, respectively). The mRNA level of collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 in miR-223 group were significantly lower than that in model group and miR-223-NC group (all P<0.05). The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA in miR-223 group was significantly lower than model group and miR-223-NC group (all P<0.05). Twist1, TGFBR2 3′UTR wild-type double luciferase reporter plasmids and miR-223 mimics were co-transfected in 293T cells, and luciferase activity was significantly reduced (0.48±0.06 vs 0.92±0.17 and 0.51±0.07 vs 0.94±0.12).Conclusion MiR-223 may inhibit the activation of fibrosis-related signaling pathway in cardiomyocytes by down-regulating the expression of Twist1 andTGFBR2 genes.