克山病microRNA差异表达谱及心肌损伤标志物研究

Keshan Disease(KD) is an endemic cardiomyopathy that the real etiology and pathogenesis are unknown, as well as the specific diagnostic indicators are unavailable. In addition, the differential diagnosis is being made difficultly between KD and dilated cardiomyopathy. This study was designed to screen microRNA gene expression profiling and exploit the diagnostic value of microRNA and its targeting gene for different patients with KD. Using microarray combined with real-time quantitative PCR method, to test the feasibility of specific miRNA and miRNA panel as a higher sensitivity and specificity diagnostic biomarker for myocardial injury associated with KD. It will contribute to the detection, the diagnosis and the intervention of patients with KD at an early stage by the development of this study.

我国克山病发病机制不清楚，病因至今未明，临床诊断尚无特异的生物学指标，以致诊断依据长期不明确，且慢型克山病和扩张型心肌病的鉴别诊断较为困难。本研究旨在利用microRNA芯片技术检测不同时期确诊的克山病病例表达谱的差异，联合实时荧光定量PCR方法确定针对克山病患者的有诊断价值的microRNA及其靶基因，并对其进行验证研究，探索特定microRNA或microRNA panel作为克山病心肌损伤生物标志物的可行性，通过项目的开展将有助于克山病病人的早期发现，早期诊断，早期干预治疗。

克山病发病机制不明，病因至今未有定论，临床诊断尚无特异的生物学标志物，以致诊断依据不明确，且慢型克山病和扩张型心肌病鉴别诊断较为困难。本项目首先通过miRNA测序技术对10名历史确诊克山病病例、10名新发克山病患者、10名扩张型心肌病患者和10名健康对照血清中miRNA表达谱进行全基因组检测，并在四组间进行两两比较，确定克山病miRNA表达谱，探索不同组间表达谱异同；进一步筛选起关键调控作用的差异表达的miRNA，再通过扩大样本例数至每组40例应用qRT-PCR技术对差异表达的miRNA进行分子生物学验证，通过这种方法确认不同时期确诊的克山病病例、扩心病病例与健康对照人群血清中测序结果显示的8种miRNA（1种miRNA被检测出两次）表达水平的差异，最终结果显示3种miRNA分别于三组间有统计学差异；采用多元logistic回归分析方法将差异miRNA引入回归方程，构建microRNA panel，并通过ROC曲线分析法评估各特异miRNA单独或联合后对克山病或扩心病诊断的灵敏度和特异度，确定对扩心病有诊断价值的1种miRNA；对克山病诊断的1种microRNA panel，确定扩心病和克山病心肌损伤相关的miRNA为miR-487b-3p、miR-6735-3p和miR-450b-5p，初步确定靶基因TM2D3基因。