miR-30c调控PAI-1在糖尿病下肢缺血血管生成中的作用机制研究

Abnormalities of angiogenesis may contribute in the pathogenesis of diabetes complications. Previous studies have confirmed plasminogen activator inhibitor-1 (PAI-1), the primary endogenous inhibitor of tissue-type plasminogen activator, as being closely associated with diabetic vascular diseases, but the molecular mechanism remains unclear. Our preliminary results indicated that PAI-1 inhibited angiogenesis via suppressing the activation of VEGFR2. Inhibition of PAI-1 by specific inhibitor increased the blood flow perfusion in a mouse diabetic hindlimb ischemia model. Furthermore, we also demonstrated that miR-30c directly targeted the 3’ UTR of PAI-1 and regulated its expression using a dual-luciferase reporter assay. Importantly, we found high glucose-induced reciprocal changes in increased expression levels of miR-30c and decreased mRNA levels of PAI-1 in human umbilical vein endothelial cells. The central hypothesis for the proposed studies is that miR-30c regulates the diabetic angiogenesis via VEGF relevant signaling pathways by targeting PAI-1. In this study, immunoprecipitation, RNA interference and hindlimb ischemia model will be used to investigate the effects of miR-30c targeting PAI-1 on VEGF/VEGFR-2 signaling and VEGF/VEGFR-2-induced angiogenesis in vitro and in vivo. Based on these findings, we hypothesize the following aims: (1) Define the effects of miR-30c on VEGF/VEGFR-2 signaling and VEGF-induced angiogenesis by targeting PAI-1; (2) Study the role and mechanism of miR-30c in the regulation of angiogenesis following hindlimb ischemia in diabetic mice and knockout mice. We anticipate that these studies will lead to the development of new strategies to control diabetic hindlimb ischemia vascular diseases.

血管生成障碍是糖尿病一个重要的继发改变。研究证实PAI-1作为纤溶酶原激活物的主要抑制物，与糖尿病血管病变密切相关，但其机制远未阐明。我们前期研究发现：PAI-1通过降低VEGFR-2磷酸化减少血管形成，抑制PAI-1增加糖尿病下肢缺血血流灌注；通过双荧光素酶报告基因实验发现miR-30c直接靶向调控PAI-1。进一步研究显示高糖导致血管内皮细胞miR-30c表达显著降低和PAI-1水平明显升高。提示miR-30c可能通过调控PAI-1影响VEGF相关信号通路参与糖尿病血管生成。因此，本项目拟从分子、细胞和整体水平采用免疫共沉淀、miRNA转染、下肢缺血模型等手段查明：1）miR-30c调控PAI-1对VEGF/VEGFR-2信号和VEGF介导血管生成的影响；2）应用糖尿病小鼠模型和基因敲除鼠，研究miR-30c调节糖尿病下肢缺血血管生成的机制，为治疗糖尿病下肢血管病变提供新的治疗靶点。

糖尿病下肢血管病变不仅导致肢端严重缺血，而且伴有血管生成障碍。VEGF信号的激活抵抗是糖尿病血管生成障碍的重要原因，但其机制仍未阐明。我们前期研究发现，PAI-1通过降低VEGFR-2磷酸化减少血管形成，抑制PAI-1增加糖尿病下肢缺血血流灌注；通过双荧光素酶报告基因实验发现miR-30c直接靶向调控PAI-1。进一步研究显示高糖导致血管内皮细胞miR-30c表达显著降低和PAI-1水平明显升高。据此我们提出假设：miR-30c可能通过调控PAI-1影响VEGF相关信号通路参与糖尿病血管生成。本项目在前期研究基础上，利用血管内皮细胞及糖尿病小鼠模型和基因敲除鼠，采用miRNA转染、激光多普勒成像、免疫共沉淀等技术，从分子、细胞和整体水平完成了预期目标，研究发现：1）体外、体内过表达miR-30c能够显著抑制靶基因PAI-1 mRNA和蛋白表达进而增加VEGF诱导的VEGFR-2磷酸化，从而增强下游的PI3K/Akt和MAPK/ERK磷酸化水平，增加VEGF介导的VEGFR-2- avβ3复合物的形成。反之，抑制miR-30c则明显增强PAI-1 mRNA和蛋白表达，进而降低VEGF诱导的VEGFR-2磷酸化及其下游PI3K/Akt和MAPK/ERK磷酸化水平，降低VEGF介导VEGFR-2- avβ3复合物形成；2）增强miR-30c或者PAI-1基因敲除或PAI-1基因抑制组均能明显促进糖尿病缺血侧下肢血流灌注改善；抑制miR-30c或增强PAI-1则显著降低缺血下肢血流灌注；3）抑制miR-30c后可明显抑制糖尿病缺血区血管生成，增强miR-30c和敲除或抑制PAI-1能够明显促进糖尿病缺血区血管生成。本课题着重关注miRNA与糖尿病血管病变的关系，首次揭示了miR-30c 调控PAI-1 对糖尿病血管病变的修复作用，为更为有效的预防和控制糖尿病血管病变症的发生提供有效的新型靶标和理论依据。