Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran, Iran.
Research Center for Advanced Technologies in Cardiovascular Medicine, Cardiovascular Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
Sci Rep. 2021 Jan 28;11(1):2548. doi: 10.1038/s41598-021-82226-z.
Cardiac fibrosis in the failing heart is modulated by activated myofibroblasts, and is a pathology marked by their deposition of extracellular matrix proteins. The TGFβ signaling pathway is important in stimulating fibrosis and therefore seems an attractive new target for anti-fibrotic therapy. The relationship between ncRNAs and TGFβ signaling pathway has been extensively studied. Here, we have provided several lines of evidence to prove that the fibrosis process could be regulated by miR-331 through targeting TGFβ signaling. First, bioinformatics analysis and dual luciferase assay validated a direct interaction between the miR-331 and TGFβ-R1 3'UTR sequence which results in the downregulation of TGFβ signaling pathway. Second, miR-331 expression was inversely related to the expression of a number of genes which are involved in extracellular matrix (ECM) production and deposition processes, both in the in vivo and in vitro fibrosis models. Third, in cultured mouse and human cardiac myofibroblasts (CMyoFbs) under ISO treatment, overexpression of miR-331 decreased the expression level of fibrosis-related genes. Consistently, western blot analysis confirmed that miR-331 overexpression ended in both Smad3 and Col1A1 protein level reduction in mouse cardiac myofibroblasts. Finally, flow cytometry analysis, cyclin D1 and D2 gene expression analysis, and wound-healing assay confirmed the inhibitory effect of miR-331 against cell proliferation and migration in ISO-treated cardiac myofibroblasts. Taken together, accumulative results showed that miR-331 reduced the level of fibrosis-related proteins in cardiac myofibroblasts culture via regulating TGFβ signaling pathway.
衰竭心脏中的心肌成纤维细胞激活可调节心脏纤维化，而心肌成纤维细胞的细胞外基质蛋白沉积是其病理学标志。TGFβ 信号通路在刺激纤维化中起重要作用，因此似乎是抗纤维化治疗的一个有吸引力的新靶点。ncRNAs 与 TGFβ 信号通路的关系已得到广泛研究。在这里，我们提供了几条证据证明，miR-331 可通过靶向 TGFβ 信号通路来调节纤维化过程。首先，生物信息学分析和双荧光素酶报告基因实验验证了 miR-331 与 TGFβ-R1 3'UTR 序列的直接相互作用，导致 TGFβ 信号通路的下调。其次，miR-331 的表达与许多参与细胞外基质 (ECM) 产生和沉积过程的基因的表达呈负相关，无论是在体内还是体外纤维化模型中。第三，在 ISO 处理的培养的小鼠和人心肌成纤维细胞 (CMyoFbs) 中，miR-331 的过表达降低了纤维化相关基因的表达水平。一致地，Western blot 分析证实，miR-331 的过表达导致小鼠心肌成纤维细胞中 Smad3 和 Col1A1 蛋白水平降低。最后，流式细胞术分析、周期蛋白 D1 和 D2 基因表达分析和划痕愈合试验证实 miR-331 抑制 ISO 处理的心肌成纤维细胞中的细胞增殖和迁移。总之，累积的结果表明，miR-331 通过调节 TGFβ 信号通路降低心肌成纤维细胞培养物中纤维化相关蛋白的水平。
