SP1介导HIF-1α调控Robo4在糖尿病视网膜病变中的作用及机制研究

Robo4 attracts great attentions in the research of diabetic retinopathy (DR), due to its function on vascular permeability and neovascularization. Recent studies show that Robo4 could facilitate the occurrence and development of DR, but its role in retinal microvascular abnormalities and the relative mechanisms are still unknown. In the previous research, we found for the first time that HIF-1α could up-regulate the expression of Robo4 through an indirect way, and PROMOTER 2.0 software also predicts SP1 exists regulatory relationships with HIF-1α and Robo4 respectively. Therefore, we put forward the hypothesis that HIF-1α can activate SP1 to regulate the expression of Robo4, which aggravates the dysfunction of retinal vessels and then promotes the progress of DR. On the basis of previous work, we will use human retinal endothelial cells and retinal pigment epithelium cells to investigate the exact regulation mechanism among HIF-1α、SP1 and Robo4, which will also be verified in the diabetic animal model. Furthermore, the influence of HIF-1α、SP1 and Robo4 on functional changes in retinal vessel is explored during the occurrence and development of DR. These studies would give useful evidences to reveal a new molecular pathogenesis of retinal vascular dysfunction in DR, as well as providing the profound basis and therapeutic way for early DR intervention.

Robo4因具有调节血管通透性、促进新生血管形成等作用而在糖尿病视网膜病变（DR）的研究中日益受到重视。已有研究显示，Robo4可影响DR的发生与发展，但其对视网膜微血管改变的作用及机制尚不清楚。在Robo4表达调控机制的研究中，我们发现HIF-1α可通过某种途径正向调节Robo4的表达，且预测分析显示SP1与HIF-1α、Robo4均可能存在调控关系。由此推测：HIF-1α通过激活SP1实现对Robo4的调控，从而加重视网膜微血管的异常改变，促进DR的发生发展。本研究拟采用不同种类的视网膜细胞深入探讨HIF-1α、SP1、Robo4三者间的调控关系和机制，并于动物模型中检测在DR发生发展过程中三者间调控机制是否存在，及该机制对视网膜微血管结构和功能的影响。本课题旨在进一步拓展对糖尿病视网膜病变发病分子机制的认识，为早期干预DR提供新的理论依据和研究方向。

以往研究表明，在糖尿病视网膜病变（DR）的发生发展中，HIF-1α与Robo4的表达水平有所升高，可加速DR的进展。为明确HIF-1α与Robo4间的调控关系，本项目基于前期实验结果和软件预测分析提出了HIF-1α/SP1/Robo4这一信号通路，并于细胞和动物水平验证了该通路的存在，检测了该通路的具体调控机制及其对视网膜细胞功能的影响。研究结果显示，HIF-1α/SP1/Robo4信号通路在高糖或低氧条件培养的视网膜细胞中和糖尿病大鼠的视网膜组织内表达升高，且抑制HIF-1α的表达可明显降低SP1与Robo4的表达水平。进一步研究三者的作用关系发现，SP1与Robo4有直接作用位点，高糖条件可诱导Robo4启动子中SP1的作用位点增多，使其与转录因子SP1结合活性增强，进而促进Robo4的转录。同时miRNA也参与该通路的调控，高糖环境下miR-125b-5p可直接靶向调控RPE细胞中SP1和Robo4的表达水平，低氧环境下miR-146a-5p可直接靶向调控RPE细胞中HIF-1α和Robo4的表达水平。通过转染siRNA或miRNA mimic的方法抑制HIF-1α/SP1/Robo4信号通路表达后检测视网膜细胞功能的改变，发现HIF-1α/SP1/Robo4表达降低可显著改善高糖或低氧诱导的视网膜细胞功能异常。由此得出结论，在DR病程中HIF-1α/SP1/Robo4信号通路被活化，加重了视网膜细胞及血管功能的异常，抑制HIF-1α/SP1/Robo4表达可有效控制DR的发展进程。本研究结果可为临床早期干预和治疗DR提供新的理论基础及方向，对DR的临床治疗具有一定的理论指导意义。