SYK相关microRNA与缺血性脑卒中发生的关联性研究及动物模型机制探讨

Recent studies have demonstrated that the gene spleen tyrosine kinase (SYK) is involved in the development of atherosclerosis, and the polymorphism of SYK gene is significantly associated with risk of ischemic stroke. microRNA (miRNA) can post-transcriptionally regulate gene expressions. However, the expression of miRNAs that might regulate SYK gene and their biological function in ischemic stroke has not been documented. In our preliminary study, the LNATM miRNA expression chip was applied to detect miRNA expression profiles of 24 ischemic stroke patients and 8 age-sex matched controls in whole blood. We found 207 differentially expressed miRNAs between the two groups, among which 13 miRNAs were predicted to target SYK gene. In this study, we will further validate the 13 miRNAs in two independent case-control populations and explore the associations between these miRNAs and the onset of ischemic stroke. In vitro cell culture will be applied to verify the miRNAs that might directly target SYK gene, and in vivo experiments using mouse model with atherosclerosis are used to demonstrate the effect of miRNAs on the development and progression of atherosclerosis. Our study will provide novel evidence for the function of miRNAs and SYK gene in ischemic stroke and strategy for disease control.

脾酪氨酸激酶（Spleen tyrosine kinase，SYK）与动脉粥样硬化关系密切，SYK基因多态与缺血性脑卒中发病风险显著相关。microRNA（miRNA）可在转录后水平调控基因表达。然而，SYK相关miRNA与缺血性脑卒中之间的关系，及其是否参与脑卒中发病机制未知。本研究前期采用miRNA表达谱芯片检测了24名缺血性脑卒中患者和8名年龄性别匹配的对照人群外周血miRNA表达谱情况，发现207个差异表达的miRNA，其中13个经生物信息学预测可能靶向调控SYK基因。因此，我们拟采用“两阶段病例对照研究”策略，探索miRNA表达水平与缺血性脑卒中发生之间的关系。细胞实验鉴定miRNA与SYK基因的靶向调节关系，并将目标miRNA转染入动脉粥样硬化（AS）小鼠模型观察其是否影响AS的发生发展。本研究将为阐明miRNA与SYK基因在缺血性脑卒中发生发展中的作用及疾病防治提供新的思路。

本课题组前期研究新发现SYK基因是缺血性脑卒中的发病易感基因，然而，SYK基因在缺血性脑卒中疾病中的调控作用尚未知。本研究旨在鉴定SYK调控相关miRNA，并分析其与缺血性脑卒中发病的关联。首先我们采用miRNA表达谱芯片和生物信息学的方法预测与SYK基因有潜在结合位点的miRNA。通过miRNA细胞转染实验，采用qRT-PCR和免疫印迹方法检测SYK基因的表达情况；采用荧光素酶报告基因实验鉴定miRNA是否能靶向结合SYK基因；人群研究中，我们在270名新发缺血性脑卒中病例和270名健康对照人群外周血中检测了目标miRNA的表达情况；采用APOE基因敲除小鼠作为动脉粥样硬化模型，腺病毒构建载体通过尾静脉注射干预的方法分析miRNA和SYK基因在动脉粥样硬化进程中的作用，并同时采用HE染色、油红O染色和免疫组化的方法探讨作用机制。结果表明，miR-129-2-3p和miR-377可能是SYK基因的靶基因。缺血性脑卒中病例外周血miR-129-2-3p表达水平显著低于对照组，且与发病风险呈显著关联性（OR: 0.88; 95% CI: 0.80-0.98; P = 0.021）。缺血性脑卒中病例外周血细胞中SYK基因表达水平高于对照组，且miR-377水平与血小板平均体积呈负相关关系。除此之外，动物实验结果表明，SYK高表达情况下促进了动脉粥样硬化进程，miR-377可通过负调控SYK基因的表达从而减缓动脉粥样硬化的发生发展。综上，本研究表明，miR-129-2-3p可能通过调控SYK基因的表达或血小板的正常功能，从而影响缺血性脑卒中的发病风险。同时，miR-377通过调控SYK基因从而影响动脉粥样硬化进程是本研究的另一个新发现。