miR-539-5p在CXCR7介导的内皮细胞间质变（EndMT）及脉络膜纤维化中的作用及机制研究

Choroidal neovascularization (CNV) is most common in age related macular degeneration (AMD) and pathologic myopia, which ultimately lead to irreversible blindness. Although anti-vascular endothelial growth factor (anti-VEGF) therapy has become a standard treatment for CNV, unsuccessful treatment outcomes have often been attributed to the progression of fibrous plaque/disciform scar. Cell transdifferentiation has been confirmed as an important cause of organ fibrosis. In our previous study, we found that endothelial-derived cells contributed to subretinal fibrosis formation through endothelial to mesenchymal transition (EndMT) and CXCR7 silencing significantly decreased Endo-MT induced by transforming growth factor-β1 (TGF-β1), but the exact mechanism remains unclear. This project aims to explore the regulatory effects of miR-539-5p target genes CXCR7, ZEB1 and Smad2 on the expression of miR-539-5p, and the regulation of target genes among each other by using the means of gene silencing and over-expressing intervention on human choroid endothelial cell (HCEC) in vitro. Moreover, we will elucidate the mechanism of CXCR7 regulates EndMT is based on miR-539-5p-mediated competing endogenous RNA (ceRNA) network. In vivo experiments, an animal model of CNV was induced by laser photocagulation, and then a variety of molecular biology methods will be use to explore the role of CXCR7 knocked out and miR-539-5p over-expression on the formation of fibrosis in CNV. We hope that these achievements can contribute to the theory and practical application for the development of new drugs on blindness caused by fibrosis in CNV.

脉络膜新生血管（CNV）最常见于年龄相关性黄斑变性和病理性近视，是致盲的直接原因。新近发现细胞转分化是器官纤维化的重要成因，我们前期研究发现脉络膜血管内皮细胞间质转化（EndMT）参与脉络膜纤维化的形成，而CXCR7基因沉默能抑制TGF-β1诱导的EndMT，但具体机制尚不明确。本课题拟以人脉络膜血管内皮细胞为研究对象，通过基因沉默、过表达技术及分子生物学手段，全面探索miR-539-5p的靶基因CXCR7、ZEB1和Smad2对miR-539-5p表达水平的影响以及靶基因间的相互调控，阐明CXCR7对EndMT的影响是基于miR-539-5p介导的竞争性内源性RNA(ceRNA)网络调控机制。我们将进一步采用激光诱导的CNV动物模型，明确CXCR7基因敲除及miR-539-5p过表达对CNV纤维化形成的作用。我们旨在通过本项研究为靶向治疗CNV导致的脉络膜纤维化打下坚实的理论基础。

项目背景：脉络膜新生血管（CNV）晚期发生的脉络膜组织纤维化将导致感光细胞死亡，是致盲的直接原因之一。然而，人们对该纤维化过程的具体机制知之甚少。新近发现细胞转分化是器官纤维化的重要成因。我们前期发现脉络膜血管内皮细胞间质转化（EndMT）参与脉络膜纤维化的形成，而CXCR7基因沉默能抑制TGF-β1诱导的EndMT。在本项目中，我们着重探讨了CXCR7在EndMT和CNV纤维化中的作用及分子机制。.研究方法：采用人视网膜微血管内皮细胞（HRMECs），通过转染CXCR7、ZEB1和Smad2 3’-UTR质粒或小干扰RNA改变其表达，观察三者相互之间的调控作用；通过转染miRNA模拟物调控miR-539-5p的表达观察miR-539-5p在TGF-β1诱导的EndMT中的作用；通过定量PCR、蛋白免疫印迹法检测miR-539-5p、CXCR7、内皮和间质细胞标记蛋白表达，以及细胞的形态变化。构建激光诱导的CNV小鼠模型，采用眼底荧光血管照影、脉络膜铺片和免疫荧光染色等方法观察野生型小鼠、miR-539-5p过表达小鼠（玻璃体腔注射腺相关病毒AAV2-miR-539-5p）和CXCR7基因敲除小鼠激光损伤区纤维化情况。.研究结果：体外研究证实：CXCR7 3’-UTR过表达能促进ZEB1和Smad2转录活性和蛋白的表达，而CXCR7基因敲除能抑制ZEB1和Smad2的表达，敲除Dicer酶能抑制上述的调控作用。同时，我们还发现TGF-β1刺激能促进EndMT的发生和抑制miR-539-5p的表达，转染miR-539-5p mimic能抑制TGF-β1诱导的EndMT现象。体内研究证实：激光诱导CNV形成第35天，miR-539-5p过表达小鼠和CXCR7基因敲除小鼠CNV损伤区Collagen I较对照组显著降低。PCR和Western blot结果同样证实miR-539-5p过表达小鼠和CXCR7基因敲除小鼠RPE/脉络膜/巩膜复合组织中间质细胞标记物表达较对照小鼠显著降低。.研究结论及科学意义：CXCR7、ZEB1、Smad2间接存在ceRNA关系，且依赖于miR-539-5p的表达，过表达miR-539-5p或降低CXCR7表达能有效抑制EndMT以及脉络膜纤维化的发生，为临床“抗新生血管”单一治疗模式向“抗新生血管+抗纤维化”联合治疗模式提供前期理论依据。