人外周血microRNA表达水平与缺血性脑卒中发生的关联性及其机制研究

MicroRNA（miRNA）can posttranscriptionally regulate the expression of genes and have critically biological functions. Circulating miRNAs often show distinct expression profiles in the disease conditions and have been validated to be the novel disease-related biomarkers. However, the association between circulating miRNAs and the onset of ischemic stroke (IS) has not been documented. In our preliminary study, the LNATM miRNA expression chip was applied to detect the miRNA expression profiles of 24 IS patients and eight age-sex matched control subjects in whole blood. We found 207 miRNAs were differentially expressed between the two groups. miRNAs with the largest fold changes were selected and validated in the same discovery cohort by real time PCR. Results suggested that nine miRNAs showed significantly differential expressions. After a thorough review of the literature, we found that most of the nine miRNAs were also related to the pathomechanisms of cardiovascular disease. In the context of that, we will further validate the nine miRNAs in two independent case-control populations and explore the associations between these miRNAs and the onset of IS. The diagnostic values of these miRNAs will also be evaluated. In addition, the target genes of miRNAs will be predicted by bioinformatics tools and validated by functional experiments. Our study may not only provide novel biomarkers for the screening and clinical diagnosis of IS，but also provide additional evidence showing the role of miRNAs in the development of IS.

MicroRNA（miRNA）可在转录后水平调控基因表达，具有十分重要的生物学功能。疾病状态下外周血循环miRNA可具特征性表达谱,与疾病的发生发展有关。然而，外周血循环miRNA与缺血性脑卒中（IS）的发生是否存在关联尚未知。本研究前期利用miRNA表达谱芯片检测了24名IS病例和8名年龄性别相匹配的对照外周血miRNA表达谱，发现207个miRNA存在显著差异。选择差异倍数较高的miRNA采用实时荧光定量PCR方法验证，结果显示9个miRNA验证成功。这些miRNA大都与心血管疾病发生发展有关。鉴于此，我们拟采用“两阶段病例-对照研究”策略，探索这些miRNA表达水平与IS发生之间的关联以及作为IS诊断标记物的临床价值,并结合生物信息学软件预测和功能实验鉴定miRNA靶基因。本研究将为IS的预防筛查和临床诊断提供新的生物标记物，并为探明miRNA在IS发生发展中的作用提供新的线索。

microRNA（miRNA）可在转录后水平调控基因的表达，具有十分重要的生物学功能。疾病状态下外周血循环miRNA可具特征性表达谱,与疾病的发生发展有关。然而，外周血循环miRNA与缺血性脑卒中（IS）的发生是否存在关联尚未知。本研究前期利用miRNA表达谱芯片检测了25名IS病例和25名年龄性别相匹配的对照人群外周血miRNA表达谱，发现389个miRNA存在显著差异。我们根据差异倍数选择了5个miRNA，采用实时荧光定量PCR的方法分两个阶段进行下一步验证。验证阶段I包括44对病例对照，验证阶段II包括302对病例对照人群。结果显示let-7e-5p和miR-363-5p在病例组中表达水平都显著高于对照组人群。let-7e-5p表达水平增加与缺血性脑卒中发病风险上升具有显著关联[校正后OR为1.89（95%CI, 1.61~2.21, p<0.001）]。采用ROC曲线和重分类分析法检验miRNA对诊断疾病的准确性。结果表明，在传统危险因素模型基础上，let-7e-5p可显著增加曲线下面积（p<0.05）。let-7e-5p的净再分类指数为16.76（11.16~22.37），综合判别指数为0.10（0.08~0.13）。采用生物信息学软件预测结合细胞实验，我们发现ELK4、CASP3、NLK和TP53基因可能受到let-7e-5p的调控，并且CASP3和NLK两个基因在病例组中显著低于对照组。. miR-363-5p表达水平增加与缺血性脑卒中发病风险上升之间也有显著关联[校正后OR为1.35（95%CI, 1.18~1.54，p<0.001）]。miR-363-5p与血浆同型半胱氨酸水平呈 正相关关系（r=0.179, p=0.011）。通过生物信息学软件预测和细胞实验验证，我们发现TIMP-1是miR-363-5p的靶基因，并且同型半胱氨酸可通过miR-363-5p下调脂多糖诱导的TIMP-1表达。.总之，本项目发现两个miRNA的表达水平与缺血性脑卒中的发病风险有关。let-7e-5p可能通过调节MAPK信号通路上一系列基因的表达从而发挥生物学作用，而miR-363-5p可能通过调控TIMP-1的表达从而参与调控同型半胱氨酸诱导的MMPs/TIMPs动态平衡，从而参与了缺血性脑卒中的发病机制。本研究可为探明miRNA在IS发生发展中的作用提供新的线索。