冠心病心外膜脂肪组织中差异性microRNAs调控apM1基因表达及其机制研究

Epicardial adipose tissue (EAT) and adiponectin(APN) are closely related to the initial and development of coronary atherosclerotic heart disease (CAD). Decreased APN was observed in EAT with CAD patients, but the mechanism has not been elaborated. MicroRNA(miRNA) are endogenous approximately 23 nucleotide noncoding RNA molecules that play important gene-regulatory roles in diverse regulation pathways and regulates the expression of approximately 30% of human protein-coding mRNAs at post-transcriptional level. MiRNAs likely play a key role in the regulation of APN expression, special production of apM1 gene, with unclear mechanism. We hypothesized that distinctive dysregulation miRNAs in EAT with CAD involved in coronary atherosclerosis process through regulated apM1 gene expression followed by our former research as well as EAT pathophysiology coupled with molecular mechanism of miRNAs. In this study, we used CAD SD rats model as well as adipose cell to study the apM1 gene expression and reason for decreased APN by distinctive miRNAs through microarray, real time PCR, gene transfection, western-blot, ELISA and fluorescence resonance energy transfer(FRET) etc. in vivo and in vitro, and furnished new idea plus novel target for intervening coronary atherosclerosis.

心外膜脂肪(EAT)、脂联素（APN）与冠心病发生发展密切相关，冠心病患者EAT 中APN减少，其确切机制不明。MicroRNAs(miRNAs)通过转录后靶向mRNA调节人体超过1/3的编码蛋白质，可能对EAT中APN表达发挥重要作用，但目前二者之间关系及调控机制研究甚少。我们提出假说：冠心病EAT中存在差异性miRNAs，调控apM1(apM1特异性表达产物为APN)，影响APN表达，对冠心病进程产生影响。本研究以SD大鼠为研究对象，应用miRNAs芯片、生物信息学、基因过表达及抑制、RT-PCR、western-blot、FRET能量转移技术等分子生物学技术，通过体内、体外两部分实验，应用上调和抑制miRNAs来分析验证其对apM1表达的调控作用，旨在明确apM1在冠心病 EAT中表达调控的重要途径，为研究冠心病发病机制提供新思路，为寻找治疗干预的新靶点提供实验依据。

本课题组前期研究显示：冠心病患者心外膜脂肪中APN降低、TNF-α等炎症因子分泌增加，局部巨噬细胞增多，心外膜脂肪厚度增加，局部处于内分泌紊乱、炎症状态，冠心病患者血清及心外膜脂肪中 APN水平较非冠心病患者降低；细胞学研究表明在炎症、胰岛素抵抗状态下3T3-L1脂肪细胞APN表达降低、TNF-α等炎症因子分泌增加。后续研究发现，兔冠状动脉粥样硬化硬化模型心包腔注射转染apMl基因，显著增加其冠心病心外膜脂肪及心包腔液中APN水平，抑制冠状动脉内膜及中膜增生。据此，本项目以翻译后修饰的视角，结合EAT病理生理特性及miRNAs分子特点，从在体、动物及细胞模型三层面进一步探讨冠心病心外膜脂肪组织中差异性miRNAs调控apM1从而影响APN表达，对冠状动脉粥祥硬化进程产生影响。通过复制冠状动脉粥样硬化SD大鼠模型，获取正常对照组、冠心病组心外膜脂肪，釆用miRNAs芯片检测CAD组及正常对照组EAT，筛选差异性miRNAs并验证；在脂肪细胞中研究差异性显著的miRNAs对脂肪细胞apM1表达影响及其机制；通过应用pre-miRNAs及anti-miRNAs，观察miRNAs对心外膜脂肪中apM1基因及APN影响 。完善apM1调控分子机制，分析冠心病患者心外膜脂肪中APN减少原因，为研发抗冠状动脉粥样硬化新药开拓新的治疗靶点。