miR-195、miR-107和miR-137的精神分裂症标志物筛查及分子调控机制研究

Our previous studies have found that several candidate genes of schizophrenia identified in the Han Chinese population, such as CACNA1C, were confirmed to be the target genes of microRNA137 (miR-137) which surpassed the genome-wide significance threshold, and miR-195, miR-107 and miR-137 were also evidenced to be abnormally expressed in some brain tissues of schizophrenic patients.Accordingly, we propose to systematically study the molecular mechanism of the 3 circulating miRNAs at posttranscriptional level in the etiology and pathogenesis of schizophrenia to examine their potential of reflecting schizophrenia disease status. Firstly, we will predict these miRNA target genes by bioinformatic tools, and then selected genes will be collectively subjected to enriched pathway analysis to see whether some key pathway patterns are regulated by these miRNAs signature. Secondly, we will experimentally test and verify target genes within the key pathway patterns regulated by these miRNAs signature by biological method in HEK293 and SK-N-BE cell lines, such as report gene, etc. Finally， we will perform genetic association analysis of these target genes in a large population，and furthermore, together with the known pathway, we will elucidate the molecular mechanism of the miRNAs signature-targets-pathway in the etiology and pathogenesis of schizophrenia. In summary, our research may help to provide additional experimental evidence for forensic psychiatry, understand the mechanism of miRNAs implicated in schizophrenia, and offer new clues and valuable insights into the molecular basis of the disease.

我们前期鉴定的CACNA1C等精神分裂症候选基因已证实是具有全基因组关联水平的miR-137的靶基因，且miR-195、miR-107和miR-137被证实在精神分裂症患者脑组织中有异常表达。本项目拟筛选这3个miRNAs在疾病外周血的分子标志物并阐明它们在疾病中作用机制。首先，运用生物信息学方法预测这3个miRNAs的靶基因并进行富集分析以构建调控网络，从而筛选这些miRNAs调控的关键通路。其次，利用报告基因等方法对这些miRNAs调控的关键通路中的靶基因进行生物学验证。最后，对这些miRNAs有明确调控作用的靶基因进行群体遗传关联分析，从而进一步阐明这3个miRNAs—靶基因—调控网络的初步作用机制。本研究将有助于为司法精神病鉴定提供辅助实验证据，拓展对miRNAs作用机制的认识，为精神分裂症分子发病机制研究提供新线索和新思路。

精神分裂症（schizophrenia, SCZ）是一种复杂的精神疾病，然而SCZ的神经生物学机制及其治疗手段尚不明确。miRNA所参与的基因表达与调控被认为是神经适应性改变的分子基础, 在神经系统发育及精神疾病中具有重要作用。为研究SCZ神经生物学机制，本项目通过以精神分裂症人群为研究对象，检测miR-195、miR-107 和 miR-137 在SCZ人群外周血血清及外周血单核细胞中表达改变。同时采用miRNA高通量测序技术（miRNA-Seq）检测SCZ人群治疗前后及健康对照人群外周血中miRNA表达谱。利用生物信息学分析方法，分析并鉴定差异表达 miRNA 及其靶基因，发现参与SCZ调控的miRNA及其靶基因调控网络。本项目进一步通过构建过表达病毒载体，结合小鼠脑核团定位技术，采用 MK801诱导小鼠SCZ行为（自主活动，高架十字迷宫），在动物水平上确定let-7b在小鼠前额叶皮质过表达对SCZ行为的影响。研究发现在30个SCZ患者样本与30个对照样本之间外周血血清和单核细胞中，3个microRNAs（miR-195、miR-107和miR-137）在SCZ患者外周血血清及外周血单核细胞中均显著表达升高。通过miRNA-seq技术检测SCZ患者在治疗前后外周血中miRNA 的表达谱改变发现参与SCZ调控的miRNA，let-7b-5p, let-7i-5p, miR-144-5p, miR-363-3p。生物信息学分析其靶基因及其参与的信号通路，发现let-7b-5p, let-7i-5p, miR-144-5p, miR-363-3p在SCZ中的调控作用与神经营养因子信号通路有关。进一步研究表明，let-7b-5p在小鼠前额叶皮质中过表达能够降低MK801诱导的小鼠自主活动升高，抑制MK801诱导小鼠在高架十字闭臂停留时间，提高其在开臂停留时间，表明let-7b-5p能够降低SCZ相关行为，具有治疗SCZ的潜在作用。此外，本项目通过收集大量中国汉族SCZ患者血样，以健康人群为对照，采用全基因组关联分析，发现了位于AS3MT-CNNM2-NT5C2基因簇区域内与SCZ有显著关联的未分型位点，以及HDAC2、SLC1A2、CACNA1D等与SCZ风险基因，获得了中国汉族人群SCZ显著相关的基因多态性位点，对后续进行SCZ机制及其治疗研究具有重要的参考意义。