女性早发冠心病的血浆microRNA表达谱及其相关发病机制的探讨

Coronary artery disease (CAD) is the leading cause of human morbidity and mortality world wide. Young women, especially those who are perimenopausal or in the early post-menopausal stage, with acute coronary syndrome have 2x higher mortality compared with age-matched men. A number of common vascular-disease-related conditions are more frequent in women than in men. Women develop a more severe or different form of vascular disease than men. However, cardiovascular (CV) risk in women is notoriously underestimated by both society and physicians. This has prompted the search for novel diagnostics that can improve the identification of female individuals at risk of developing premature CAD (pCAD). Also, it is very important to do research on pathogenesis of pCAD in women (premature CAD was defined as a cardiac event before the age of 65 for females). The 100 female pCAD and 100 healthy female control subjects were already enrolled from our hosptial. This proposal seeks support to accomplish the following aims:1. pCAD-associated plasma microRNA expression profile: using microarray-based microRNA (miRNA) expression profiling and quantitative reverse transcriptase-polymerase chain reaction, we compared the miRNA expressions in plasma samples from female pCAD patients and healthy control subjects(in case-control study) to get signature plasma microRNA expression profile of pCAD in women. 2. miRNA functional study: We investigate the potential cellular origin of candiate miRNA by in situ hybridization and confirm direct target genes of candiate miRNAs by cell transfection and measurement of target genes and proteins' expression. 3. Data intergration: in case-control study, we investigate the assiocation between target genes expression(mRNA), miRNA related Single Nucleotide Polymorphisms(SNPs,located at regions encoding miRNA or miRNA binding sites), biomarkers and female pCAD. Furthermore, we integrate the information from miRNA, mRNA, SNPs and biomarkers to discuss the possible pathogenesis of female pCAD by analysing the miRNA target genes and related pathway. It will give important evidences to prevention, diagnosis, treatment of female pCAD.

女性早发冠心病（pCAD）患者通常血管病变严重，远期预后差，而临床上容易被忽视导致延误诊治，寻找对疾病准确识别及预测的敏感指标，研究疾病发生发展机制具有重要意义。项目前期已收集女性pCAD患者100例及健康对照100例,本课题拟：1、通过病例对照研究，应用microRNA(miRNA)芯片技术及定量逆转录聚合酶链反应明确女性pCAD的血浆miRNA 表达谱，寻找疾病的候选标记物；2、通过miRNA功能研究（原位杂交，细胞转染，检测基因蛋白表达）确定特征性miRNA靶基因；3、通过病例对照研究，分析靶基因表达水平（mRNA），miRNA编码/结合位点基因多态性(SNP)及相关生物标记物与疾病表型的关系，并整合miRNA-mRNA-SNPs数据，与生物标记物检测所获得的靶基因信号通路信息进行比较，探讨可能存在的女性pCAD发病机制，这些差异表达的基因和生物标记物对诊断，治疗和预后有重要意义。

女性早发冠心病（pCAD）患者通常病变程度严重，远期预后差，寻找对疾病准确识别及预测的敏感指标及研究疾病发生发展机制具有重要意义。我们首次探索miRNA作为女性早发冠心病候选标记物的可能，并进一步通过关联分析探讨可能存在的疾病的发病机制。我们首先采用TaqMan 探针miRNA低密度芯片获得13个表达有明显差异的miRNA，继之在30:30的病例对照研究中，应用实时荧光定量PCR技术进行验证，发现miRNA-99a （miR-99a）在病例组中表达明显上调；进一步通过病例对照研究，我们分析miR－99a相关SNP, 生物标记物与疾病表型的关系。我们发现miR-99a靶基因ANRIL相关的SNP: rs4977574基因型及等位基因频率在女性早发冠心病组与对照组之间的分布有显著性差异，rs4977574G等位基因明显增加发生女性早发冠心病的风险（ORs:1.95, 95%CI: 1.07,3.57）。根据miR-99a涉及炎症及代谢等相关信号通路，我们检测生物标记物，发现血尿酸及糖化血红蛋白在病例对照组间有差异，高尿酸及高糖化血红蛋白增加女性早发冠心病的可能。我们进一步研究高糖化血红蛋白在rs4977574基因型中的分布情况，发现在rs4977574GG+GA组中高糖化血红蛋白的发生率明显增加，综合SNP分布情况（ANRIL:rs4977574），与根据生物标记物(糖化血红蛋白)检测所获得的相关信号通路信息进行比较，我们推测女性pCAD发生发展可能途径之一：miR-99a通过调控ANRIL基因的表达, 参与机体代谢相关通路信号传导，导致代谢异常特别是血糖代谢异常，从而参与女性冠状动脉粥样硬化的形成。.我们同时关注女性早发冠心病危险分层的相关因素，分析发现①炎症相关生物标记物：高敏CRP和血沉的升高明显增加发生急性冠脉综合征的风险。因此推测炎症与早发冠心病危险分层相关；②焦虑抑郁及睡眠状况对疾病发生发展的影响，发现睡眠质量与疾病危险分层有关，睡眠障碍者出现急性冠脉综合征的可能性更大，睡眠障碍可能是疾病的重要危险因素。