MicroRNAs网络调控阿尔茨海默病病理机制与临床验证研究

The pathological process of Alzheimer's disease (AD) is closely related to the miRNA regulatory mechanisms. Our team has found significant differences of single set of miRNA expression in peripheral blood in different cognitive function groups. But the network control mechanism of miRNAs for AD pathological process is still unknown. We study the epigenetic regulation of miRNAs network that the target is BACE1mRNA in this project. The research will determine the intrinsic characteristics and interaction mechanisms of miRNA network on AD pathological processes and the regulation trends of different miRNAs on BACE1mRNA. The research will determine the critical path that the miRNA induce the apoptosis of human neuronal-biovol human primary cell in the microenvironment of ER stress, oxidative stress, inflammation, lack of energy and hypoxic by the techniques of RT-PCR, gene transfection, gene silencing, Western Blot and Co-immunoprecipitation and others and re-verification. We also therefore setup this study to explore the possibility if the plasma measurements are correlated with the CSF ones for miRNAs, and to examine whether each or combination of these miRNAs plasma measures is capable of especially discriminating amnesia Mild Cognitive Impairment (aMCI) from Normal control (NC), and also AD from NC, AD/aMCI from NC, or even AD from aMCI. We are especially interested in examining such feasibility in distinguishing aMCI from NC as it is relatively more difficult to diagnose aMCI in contrast to NC than to diagnose AD from NC.

阿尔茨海默病（AD）已成为严重的公共卫生问题，其发生发展与微小RNA（miRNA）调控有关。已发现不同认知功能老人外周血单一miRNA表达存在显著差异，但miRNAs网络如何影响AD病理过程仍不明。本研究针对靶目标为BACE1mRNA的miRNAs网络进行表观遗传调控研究，确定不同miRNA对BACE1mRNA的调控强度和miRNAs网络协同调控机制及其内部相互作用。模拟氧化应激、炎症、能量缺乏和缺氧微环境，利用RT-PCR、基因转染、基因静默、Western Blot及免疫共沉淀等技术确定miRNA诱导人神经元细胞-biovol人类原代细胞凋亡的关键途径并验证。与正常对照及轻度认知功能损害（aMCI）比较，探索轻度AD患者脑脊液（CSF）及外周血miRNAs表达与CSF及外周血病理标志物水平变化的关系，确定外周血miRNAs表达诊断aMCI的价值并验证。为早期干预AD的发生发展提供靶点。

阿尔茨海默病（AD）的疾病进程被认为是一个连续的疾病谱，MCI是介于临床前期和痴呆之间的中间临床状态，每年有10%-15%进展为AD。其中aMCI被认为是进展为AD的重要危险因素之一，早期识别aMCI能为治疗AD提供时间窗。特定的微小RNA（miRNA）能够调控BACE1 mRNA，来增加Aβ的生成，从而促进AD发生。AD患者的血浆和脑脊液中miRNA表达与正常人存在差异，这使miRNA作为诊断的生物标志物成为可能。研究表明，AD患者与aMCI患者血浆中miRNA表达同样存在差异。因此，AD的miRNA表达谱无法为aMCI的诊断提供参考，所以我们关注aMCI患者血浆中差异表达的miRNAs，考察其是否可作为aMCI患者的诊断标志物，同时考察其在AD的病理生理过程中的调控作用。.我们招募65例aMCI患者和55例正常对照老人（NC），分为筛选集、分析集和验证集。筛选集的aMCI患者经18F-flutemetamol PET 金标准诊断。采用microarray芯片检测两组各样本血浆中miRNA水平，发现aMCI患者血浆miRNA的差异表达谱。筛选以BACE1 mRNA作为靶点的miRNA，在分析集采用实时荧光定量PCR分别检测各样本血浆中miRNA水平，验证芯片筛查结果。结果发现与NC相比，aMCI患者血浆中miR-1185-2-3p、miR-1909-3p、miR-22-5p、miR-107和miR-134-3p显著偏低。采用验证集验证上述联合诊断模型的诊断效力，灵敏度和特异度分别为80.0%和82.5%。机制研究的结果表明，miR-1185-2-3p、miR-1909-3p、miR-22-5p和miR-107可在海马神经元中调控BACE1蛋白的表达，影响下游CTF-β蛋白产生。在细胞水平模拟不同的脑部微环境，探究脑部微环境对miRNA、BACE1 mRNA和BACE1蛋白表达的影响。结果发现轻度内质网应激和炎性环境下，BACE1mRNA和BACE1蛋白表达有明显的上调，而轻度氧化应激并不能引起BACE1表达的变化。分别过表达miR-107, miR-22-5p和miR-1185-2-3p，可使上调的BACE1蛋白表达下降，减弱轻度内质网应激和炎症效应。但探究性结果显示三种脑部微环境对miRNA的表达具有一定影响，炎症过程是AD发病的重要诱发因素。