Department of Cardiovascular Surgery; The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Department of Cardiovascular Surgery; The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Biomed Pharmacother. 2017 Nov;95:215-222. doi: 10.1016/j.biopha.2017.07.104. Epub 2017 Sep 12.
Endothelial-mesenchymal transition (EndMT) is closely associated with embryogenesis, injury restitution, tissue neogenesis, tumor progressions and viscera fibrosis. EndMT may occur in the proximal tubular endothelial cells, inducing fibroblasts to produce matrix and then accelerating the process of cardiac fibrosis. Transforming growth factor-β1 (TGF-β1), a profibrotic cytokine, was recently shown to be a crucial trigger of EndMT in tubular endothelial cells. Increasing evidence suggests that growth factor receptor-bound 2 (GRB2) dysfunction affects fibrocytes; thus, GRB2 may be a novel target for treating fibrosis. The miR-200 miRNA cluster (miR-429, miR-141, miR-200c, miR-200b and miR-200a) was reported to inhibit EndMT. However, the underlying mechanisms, specifically that of miR-200a, are unclear. To elucidate the vital role of miR-200a in EndMT, we established a cardiac interstitial fibrosis model with a widely used EndMT assay, TGF-β1-induced EndMT in human aortic endothelial cells (HAECs). We found that overexpression of miR-200a blocked EndMT in HAECs by inhibiting fibroblast-specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) expression and increasing platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (VE-cadherin) expression, regardless of the presence of TGF-β1. MiR-200a expression was suppressed during the EndMT process, in both time- and dose-dependent manners, following GRB2 upregulation. EndMT was promoted by ectopic expression of GRB2 via decreased CD31 and VE-cadherin. Furthermore, EndMT was partially inhibited by co-transfection of miR-200a with GRB2 ORF, likely by restoring CD31 and VE-cadherin expression. MiR-200a negatively regulated GRB2 protein levels via direct binding to the GRB2 3'UTR. Finally, these discoveries may provide novel insights into the functional mechanism of miR-200a in regulating fibrosis via the TGFβ1/miR-200a/GRB2/EndMT pathway, and miR-200a may serve as a new target for treating fibrosis in the future.
内皮-间质转化（EndMT）与胚胎发生、损伤修复、组织新生、肿瘤进展和内脏纤维化密切相关。EndMT 可能发生在近端肾小管内皮细胞中，诱导成纤维细胞产生基质，从而加速心脏纤维化的进程。转化生长因子-β1（TGF-β1），一种促纤维化细胞因子，最近被证明是管状内皮细胞中 EndMT 的关键触发因素。越来越多的证据表明，生长因子受体结合蛋白 2（GRB2）功能障碍影响纤维细胞；因此，GRB2 可能是治疗纤维化的新靶点。miR-200 微 RNA 簇（miR-429、miR-141、miR-200c、miR-200b 和 miR-200a）被报道能抑制 EndMT。然而，其潜在机制，特别是 miR-200a 的机制尚不清楚。为了阐明 miR-200a 在 EndMT 中的重要作用，我们建立了一种心脏间质纤维化模型，使用广泛应用的 EndMT 检测方法，即 TGF-β1 诱导的人主动脉内皮细胞（HAECs）EndMT。我们发现，miR-200a 的过表达通过抑制成纤维细胞特异性蛋白 1（FSP-1）和α-平滑肌肌动蛋白（α-SMA）的表达，增加血小板内皮细胞黏附分子 1（CD31）和血管内皮钙黏蛋白（VE-cadherin）的表达，从而阻断 HAECs 中的 EndMT，无论是否存在 TGF-β1。miR-200a 的表达在 GRB2 上调后，无论是时间依赖性还是剂量依赖性，都在 EndMT 过程中受到抑制。通过外源性表达 GRB2 促进 EndMT，导致 CD31 和 VE-cadherin 减少。此外，通过与 GRB2 ORF 共转染，miR-200a 部分抑制 EndMT，可能通过恢复 CD31 和 VE-cadherin 的表达。miR-200a 通过直接结合 GRB2 3'UTR 负调控 GRB2 蛋白水平。最后，这些发现可能为 miR-200a 通过 TGFβ1/miR-200a/GRB2/EndMT 途径调节纤维化的功能机制提供新的见解，miR-200a 可能成为未来治疗纤维化的新靶点。
