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

Abstract

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.