miR-106b对动脉粥样硬化斑块稳定性的影响及其作用机制的研究

Angiographic and pathological studies have shown that most cardiac events, such as myocardial infarction or sudden cardiac death, are related to the vulnerable plaque rupture which occurs at sites with less severe luminal stenosis from, so the vulnerability is considered to be the most important factor of a plaque for its clinical consequence. MicroRNAs (miRNAs) are ~22 nucleotides long non-coding RNAs, which negatively regulate gene expression by binding to the 3' untranslated region (UTRs) of targeted mRNAs. One miRNA can target hundreds of .mRNAs and each mRNA can be regulated by multiple miRNAs simultaneously, which forms a complex network. In previous studies, using MiRNA Low Density Array to assess total RNA isolated from plasma, we analyzed the miRNA expression profiles of patients with chest pain attributable to non–coronary atherosclerosis stenosis-related causes and unstable angina patients with vulnerable plaque. The expression of miR-106b were significantly elevated in the plasma of CAD patients with unstable plaque compared with control cases (Fold change= 23.2). A co-expression network analysis offers additional insights compared with studying deregulated individual miRNAs. So we use co-expression network analysis to evaluates changes of individual miRNAs in the context of the overall miRNA network. And we found that miR-106 was the key node in the network of patients with unstable plaque. Besides, several miRNAs related directly to miR-106b in the co-expression network were reported to be inflammation associated and might participate in the pathogenesis of atherosclerosis. So we decided to study the role of miR-106b in the pathogenesis of vulnerable plaque and its underlying mechanism. And we would further observe the influence of miR-106b on the index of plaque stability (e.g. number of smooth muscle cells, content of fibrosis fiber, cell apoptosis and size of lipid pool) and offer theoretical basis for the translation of miR-106b level manipulating to clinical application of vulnerable plaque therapy.

目前认为决定动脉粥样硬化疾病危害性的根本因素是斑块的稳定性。现有研究表明 miRNAs参与调节斑块稳定性的多个环节，且 miRNAs 相互之间构成复杂的网络调控关系。本项目组既往研究发现存在不稳定斑块的患者血浆中 miR-106b 的水平显著上调，进一步采用共表达网络分析发现miR-106b 处于两大子网络桥接的关键位置，提示miR-106b极有可能参与调节斑块的稳定性。因此本项目组拟首先确定miR-106b在斑块局部的细胞来源，进一步应用基因芯片筛查和生物信息学方法分析miR-106b潜在的靶基因，进而通过luciferase报告基因法确定miR-106b的靶基因。最终在大体动物水平通过特定调节体内miR-106b的表达水平，观察其对于斑块稳定性的影响和具体的分子机制。旨在阐明miR-106b对于斑块稳定性的具体影响效应和潜在的作用机制，为基于miRNAs的治疗策略向临床转化提供参考。

决定动脉粥样硬化疾病危害性的根本因素并不是动脉粥样硬化斑块的大小，而是动脉粥样硬化斑块的稳定性，miRNAs 极有可能在动脉粥样硬化斑块由稳定表型向易损表型转化过程中发挥着关键性的调节作用，而本项目前期研究发现miR-106b极有可能参与调节斑块的稳定性。因此，本研究首先通过细胞水平的试验确定了miR-106b在动脉粥样硬化斑块处的主要来源为血管内皮细胞；之后通过高通量基因表达谱芯片筛查过表达miR-106b前后，人脐静脉内皮细胞 HUVEC发生显著变化的mRNA表达谱，进一步通过综合分析文献回顾、miRNAs靶点数据库预测的方法最终确定miR-106b的靶基因，包括细胞凋亡的关键调节因子PTEN、动脉粥样硬化中关键炎症信号调节通路STAT3等靶基因，并在离体细胞水平通过调控miR-106b的表达水平进一步通过luciferase报告基因法将上述基因确定为miR-106b的靶基因。同时在大体动物水平，通过ApoE基因敲除、动脉套管置放等处理手段构建易损斑块小鼠模型，发现通过向小鼠注射miR-106b 的过表达试剂（agomir-106b）特异性调控miR-106b的表达水平，小鼠的主动脉处斑块局部miR-106b的靶基因表达水平显著下调，而斑块的稳定性指标（单核细胞粘附数目、巨噬细胞含量和胶原纤维含量等）显著改善。综合上述结果，本研究为探讨基于miR-106b的分子靶向治疗易损动脉粥样硬化斑块的机制奠定了坚实的工作基础，同时对于认识miR-106b在动脉粥样硬化基因表达调控网络中的核心作用提供了重要的启示性线索。项目资助发表SCI收录期刊论著3篇，待发表中文核心期刊论著1篇。协助培养博士生6名，其中3名已经取得博士学位，3名在读。项目投入经费23万元，支出20.7405万元，各项支出基本与预算相符，剩余经费2.2595万元，剩余经费计划用于本项目研究后续支出。