MiR-223在糖尿病相关缺血性脑卒中发病机制中的作用研究

Platelet activation plays an very important role in pathogenesis of ischemic stroke(IS),which is the leading cause of death in the world. CalDAG-GEFI and P2RY12 receptor (P2RY12) can both promote the activation of GPⅡb/Ⅲa, which is a key step in platelet activation and aggregation. MiR-223 is the most abundant miRNA in platelets. Our preliminary research had found that the amouts of miR-223 reduced apparently in IS patients who also suffered with Type 2 Diabetes Mellitus (T2DM) than the healthy controls. Thus, we suppose that miR-223 may play an important role in the pathogenesis of IS with T2DM by modulating the expression of CalDAG-GEFI and P2RY12. In this study, we will examine the platelet activation rate and the expression level of miR-223, P2RY12 and CalDAG-GEFI by method of real-time RCR, Western blot and flow cytometry. Besides, we will construct a recombinant plasmid which contains mir-223, P2RY12, and CalDAG-GEFI gene and transfect it into HEK293 cell, and make full attempt to find out if CalDAG-GEFI is the target gene of miR-223 by using Dual-Luciferase reporter gene assay. In addtion, we will explore whether high glucose has any influence on mir-223 expression and the methylation of its promoter, which could activate the platelets and increase the risk of CI, by studying megacaryocytes treated with high glucose,methylation reagent and hypoglycemic drugs. It will help provide new ideas and evidences on CI new drugs research.

血小板活化在缺血性脑卒中(IS)发病中起重要作用,CalDAG-GEFI和P2RY12促GPⅡb/Ⅲa活化是血小板活化的重要环节。MiR-223是血小板中高表达的miRNA,课题组前期研究发现伴糖尿病的IS患者血小板miR-223显著降低,提出miR-223表达下降可能通过增加P2RY12和CalDAG-GEFI表达,参与糖尿病相关IS的发生发展。本研究拟运用半定量Real-Time RCR、Western blot和流式细胞分析等技术,观察伴糖尿病的IS患者血小板miR-223表达、P2RY12和CalDAG-GEFI蛋白表达及血小板活化率的变化情况,并通过报告基因分析确定CalDAG-GEFI是否为miR-223的靶基因；然后通过高糖、去甲基化和降糖药处理巨核细胞,明确高糖是否通过促进miR-223启动子甲基化,引起血小板活性升高，增加IS的发病风险,从而为IS防治药物的研发提供新思路

本课题首次从参与血小板活化的通路出发，探讨血小板相关microRNA在伴有糖尿病的缺血性脑卒中患者中的表达情况以及在血小板活化中的作用，研究结果发现血小板miR-223和 miR-146a表达下调可能是血小板活化的重要分子机制，高糖可能通过下调miR-223和 miR-146a的表达，进而上调血小板活化相关靶基因的表达，从而增加缺血性脑卒中的发病风险；血小板和血浆miR-495和miR-107的表达可能与缺血性脑卒中的发病风险无明显相关性。进一步通过报告基因实验，证实了FAM5C为miR-223的靶基因，发现高糖可通过下调miR-223的表达，减弱其对靶基因FAM5C表达的抑制，从而促使其靶基因FAM5C的表达增高，进而促进动脉粥样硬化及血栓形成，增加脑梗死的发病风险。以上研究结果提示miR-223可作为新的抗血小板药物作用靶点，为糖尿病相关缺血性脑卒中患者的治疗开辟了新的途径。最后，我们将表观遗传学与脑血管病临床研究有机结合，首次应用BSP技术研究了miR-223基因启动子区甲基化与动脉粥样硬化性脑梗死(Atherosclerotic Cerebral Infarction, ACI)及动脉粥样硬化的关系，研究结果发现，miR-223基因启动子区低甲基化与ACI及颈动脉粥样硬化有关；miR-223基因启动子区的甲基化水平与血浆胆固醇浓度呈正相关，从甲基化调节的表观遗传学角度阐明了缺血性脑卒中的发病机制，研究结果对于指导缺血性脑卒中的防治、发掘新的药物作用靶点具有重要的临床意义。