与氧化脂蛋白相关的血浆特异性microRNA在动脉粥样硬化中的作用研究

MicroRNAs (miRNAs) are members of a class of small, endogenic and noncoding RNA molecules. Serum/plasma miRNAs can be detected in a stable form. It could be a potential new biomarker for cardiovascular diseases. However, the origin and mechanism of miRNA are still poorly understood. The clinical and experimental studies on the association between miRNA and the severity degree of atherosclerosis (As) have not been reported. Our previous studies found the abnormal miRNAs expression profile in the patients with coronary artery disease (CAD), and the significant correlation between the oxidized lipoproteins and miRNA. However, the mechanism is not explicit currently. Thus, we hypothesized?that abnormal miRNA expressions in CAD patients are associated with oxidized lipoproteins levels, inflammation reaction and the degree of As; miRNA may be directly released from the ruptured or unstable plaques. To verify this hypothesis, TaqMan low density array assay and a stem-loop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay are used to explore the associations of miRNA with oxidized lipoproteins, inflammation factors and the degree of As from clinical, cellular, tissular and animal studies. We want to clear some target genes and mechanism of abnormal miRNA, and to observe the intervention effect of miRNA inhibitor. This study will provide a new approach and theoretical foundation for clinical diagnosis and treatment in As.

miRNA是一种内源性，非编码的小RNA。血清/血浆miRNA稳定性高，可望成为心血管疾病诊断的新型标志物；但miRNA的来源及变化机制尚不清楚，有关miRNA与动脉粥样硬化(As)程度关系的临床和实验研究亦未见报道。我们前期研究提示冠心病患者血浆miRNA表达谱异常，且与氧化脂蛋白水平相关，但其机理尚不明确。为此，我们提出假说：冠心病患者血浆miRNA表达异常，与氧化脂蛋白水平、炎症和As程度相关；miRNA可来源于破裂、不稳定As斑块或受损细胞的直接释放。为了验证这一假说，本项目拟采取低密度芯片及实时荧光定量PCR等技术，从临床、细胞、组织以及动物整体水平探讨冠心病患者血浆特异性miRNA表达谱与氧化脂蛋白、炎症因子及As程度间关系；明确某些异常表达的miRNA作用的靶基因及其致病机制、miRNA抑制剂干预效果，为As性疾病的诊断、治疗提供新的途径和理论依据。

microRNA(miRNA)是一种内源性非编码小RNA，可望成为心血管疾病的新型标志物。本项目首先采用Taqman低密度芯片结合实时荧光定量PCR技术，筛选并鉴定了血脂异常心血管疾病危险分层人群的血清特异miRNA表达谱；通过ROC曲线分析、Logistic回归分析、Spearman相关性分析等统计学方法，准确评估了血清特异miRNA应用于血脂异常人群CVD危险分层的临床价值；发现某些特异变化的血清miRNA与其血脂指标、氧化脂蛋白、炎症因子、心肌损伤标志物及冠脉病变Gensini积分等均显著相关，不仅可反映As病变的严重程度，还与脂质代谢紊乱过程密切相关。进一步体外细胞实验，分析了氧化脂蛋白诱导巨噬细胞miRNA表达的变化， 发现泡沫细胞中某些miRNA的水平显著升高；进而通过生物信息学预测、荧光素酶报告基因实验及Western blot等手段，构建并验证了特定miRNA通过抑制胆固醇外流关键蛋白的表达、促进泡沫细胞形成的分子调控网络；并分析了特定miRNA在动脉粥样硬化发生、发展过程中发挥的具体作用。最后体内动物实验，通过构建LDLR-/-小鼠的动脉粥样硬化模型，分析了其动脉斑块和血清中特定miRNA及miRNA调控的靶蛋白、mRNA水平的变化，探讨了特定miRNA水平与氧化脂蛋白、炎症因子及As程度的关系，并观察了过表达、抑制表达特定miRNA抑制剂对As发生、发展的影响。本项目累计已发表论文23篇，其中6篇被SCI收录；多次参加国内外学术会议；获得军队医疗成果二等奖1项；为动脉粥样硬化性疾病的预测和病情评估提供了新的途径和理论依据。