血清miRNA表达谱和miRNA基因及其靶位点多态性与阿尔茨海默病的关系研究

MicroRNAs (miRNAs) are a class of endogenous, small non-coding RNAs that mediate post-transcriptional gene silencing by complementary binding to the 3' untranslated region (3'UTR) of target mRNAs. In 2008, it was firstly reported that miRNAs could stably exist in sera, with its expression profile changes in accordance with the biological and pathological conditions. We hypotheses that Alzheimer's disease(AD)-specific serum miRNAs expression profiles exist in AD patients and single nucleotide polymorphisms (SNPs) in miRNAs and miRNA targeting sites are associated with AD susceptibility by affecting miRNA-mediated AD-related gene repression. Our previous study, using the candidate miRNA strategy, have identified serum miRNA as a potential biomarker for AD diagnosis. Moreover,we have identified 70 common functional SNPs in Han Chinese population that located in human miRNA genes or miRNA binding sites of the AD-susceptible genes at the whole genomic level by bioinformatics analysis. Based on the established large sample of Han Chinese AD patients and our previous work, we will firstly determine the AD-specific serum miRNAs expression profiles in AD patients through the following three phases: primary screening, secondary screening and verification. We will also analyze and predict the screening results so as to get the related mechanisms between the specific serum miRNAs and AD pathogenesis. Secondly, we will conduct a two-stage study (primary screening and verification) to examine the impact of a panel of 70 common functional SNPs, selected in our previous work, on the risk of AD patients in Han Chinese. A comprehensive investigation on the genotype-phenotype interaction will also be conducted. Finally, the above-mentioned two parts of findings will be combined for detecting the related mechanisms between SNPs in miRNAs and miRNA targeting sites and the risk of AD by the genotype-miRNA expression interaction analysis. This study will screen out new biomarkers for AD, and provide new content for clarifying molecular mechanisms of the pathogenesis and identifying therapeutic targets for AD.

MicroRNA（miRNA）是一类非编码的小RNA分子，在基因表达的遗传学调控中发挥着重要的作用。我们前期采用候选miRNA的研究策略发现血清中miRNA可作为潜在的生物学标记用于AD的诊断，并通过生物信息学分析对全部的miRNA及AD易感基因miRNA靶位点进行了SNPs筛查，发现了70个在汉族人常见的功能性SNPs。本项目拟在已建立大样本汉族人AD资源库和前期工作的基础上，首先通过基于深度测序的初筛、复筛和验证三个阶段，全面的筛选出AD特异血清miRNAs表达差异谱，并用生物信息学预测潜在的机制；其次通过初筛和验证两个阶段，分析前期筛选的70个SNPs单独或联合与AD遗传易感的相关性，综合评价基因型-表型相互关系；最后结合前两部分的研究结果，进行基因型-表达水平分析，探讨潜在的分子机制。本项目的研究结果不仅可以推动AD的遗传学认识，而且有可能对本病的早期预警、诊断和治疗提供新的靶点。

本项目历时4年，在前期建立的汉族人阿尔茨海默病遗传资源库基础上，应用高通量二代小RNA测序、PCR-LDR、SnaPshot、QRT-PCR、生物信息学等技术，建立RNA 直接测序技术检测AD关键microRNA表达谱及AD候选基因突变的技术平台。本项目建立了一套稳定的外周血清microRNA表达谱研究体系，发现外周血清中microRNA的表达谱可以用于AD的早期预警和诊断：采用二代测序技术对AD患者外周血清中的microRNA进行全基因组深度测序，首次发现外周血清中microRNA的表达谱可以用于AD的早期预警和诊断，其中miR-342-3p的敏感性和特异性最高。此外，进一步分析基因-基因和基因型-临床表型分析，筛选出21个AD发病密切关联基因及位点，验证后进一步构建了miR-342-3p-APH1B基因-AD发病预测模型。发现APH1B的rs10519204和miR-342-3p表达水平呈正相关，表明rs10519204突变基因型可见较高的miR-342-3p表达水平,而rs10519204突变基因型TT对AD的发病起保护作用，揭示了AD发病机制的潜在分子通路。研究成果目前在《Molecular Neurobiology》、《Scientific Reports》、《Journal of Alzheimer’s Disease》、《Current Neurovascualr Research》、《中华神经科杂志》等核心期刊上正式发表论文9篇，其中SCI收录 8篇，CSCD收录 1篇，影响因子大于5分的3篇，已发表的论文被国内外同行多次引用, 其中，发表在在痴呆领域标杆期刊J Alzheimers Dis杂志的论文，发表半年内被引用17次，被杂志编委会列为2015 Alzheimer Award的候选文章之一。本项目受邀在2014年亚洲大洋洲神经病学会议、2015年日本神经病学年会、2017年美国神经病学年会做大会报告和壁报交流。项目部分成果经科技鉴定达到国际先进水平，荣获美国神经病学学会国际学者奖、山东省研究生优秀科技创新二等奖、山东省优秀硕士学位论文、青岛市科技进步二等奖等。该项目研究成果在青岛市市立医院、青岛市海慈医疗集团、青岛市中心医院、青岛市精神卫生中心等多家三甲医院推广应用，加强了阿尔茨海默病的宣教工作，提高了应用单位阿尔海默病的早期预警诊断防治水平。