孤独症相关miRNA的遗传筛选及其调控机制研究

Autism is a neurodevelopmental disorder with high heritability. The expression regulation mechanism of the autism susceptibility genes in brain remains unclear. The mechanism of temporary and spatial expression of genes is complicated. miRNA is one of the most critical molecules which regulate genes expression. Therefore, it is important to identify autism-related miRNAs and investigate their regulation mechanism. In this study, we will perform association analyses for single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) in miRNA genes and their targeted genes by utilizing genome-wide association dataset in Chinese Han autism trios. Then, the replication study will be performed in additional 500 autism trios. To discover the difference, expression level of miRNA and mRNA in the peripheral blood of 10 autism dizygotic twins will be measured by using miRNA and mRNA microarray. Further, the replication study will be performed to confirm the results. Through the genetic stuides and bioinformatics analyses, autism-related miRNAs could be identified. After that, the function of autism- and neurodevelopment-related miRNA on neurite development and behavior of mice are explored by transfecting miRNAs to neuron and the brain regions of mice. We try to explore the regulation mechanism of miRNAs and the signaling pathway of their target genes. This study will provide an insight into understanding the pathogenesis of miRNA on autism.

孤独症是一种多基因致病、高遗传度的神经发育障碍。孤独症易感基因在脑发育过程中时空表达的调控机制尚不完全清楚。miRNA是调控基因表达的重要分子，故筛选孤独症相关miRNA并研究其调控机制有重要意义。本研究拟在中国汉族孤独症核心家系中利用全基因组数据进行miRNA与靶基因单核苷酸多态性关联研究及拷贝数变异分析，并在独立样本孤独症核心家系中进行验证。此外对10对孤独症异卵双生子进行外周血miRNA和mRNA表达谱芯片检测和独立样本重复验证，拟发现孤独症患者miRNA的差异表达。利用遗传和生物信息分析，探讨miRNA对mRNA的调控，筛选出孤独症相关miRNA。针对差异表达miRNA和及其靶基因开展功能研究，探讨其对神经元轴树突发育和小鼠行为表型的影响，在细胞和整体动物水平明确与孤独症脑发育关系最为密切的若干miRNA，继而研究其调控下游靶基因通路的分子机制，为解析孤独症发病机制提供研究线索。

孤独症是一种高遗传度的神经发育障碍。miRNA是调控基因表达的重要分子，故筛选孤独症相关miRNA并探讨其调控机制有重要意义。本课题对影响miRNA结合的单核苷酸多态性位点（single nucleotide polymorphism, SNP）筛选提示SNAP25基因3'非编码区的rs8636参与孤独症发病。SNAP25基因编码的突触小体相关蛋白25kDa，是囊泡运输中的关键成分之一，在神经递质的囊泡运输、轴突生长和神经可塑性等中枢神经系统功能中具有重要作用。本研究采用小RNA测序的方法对孤独症患者和健康对照人群的血浆miRNA表达水平进行分析，结果发现共243个miRNA（76个已知miRNA和167个新预测miRNA）的表达水平在孤独症组和对照组之间存在显著差异，其中181个miRNA在病例组中显著上调，42个miRNA在病例组显著下调。在76个差异表达的已知miRNA中，hsa-miR-21-3p、hsa-miR-346、hsa-miR-486-5p和hsa-miR-92b-3p等曾被报道在孤独症患者中表达水平显著异常。此外，hsa-miR-92a-3p、hsa-miR-1-3p和hsa-miR-221-3p等在精神分裂症等神经精神疾病中表达水平显著异常。富集分析提示差异表达miRNA的靶基因在孤独症相关基因集中显著富集。在另外独立样本的孤独症与正常对照中验证发现，与健康对照组相比，孤独症患者血浆中hsa-miR-615-3p和hsa-miR-483-3p的表达水平显著上调，hsa-miR-1-3p的表达水平显著下调。以上结果提示，孤独症患者血浆miRNA表达水平可能与孤独症的发病有关，其中hsa-miR-615-3p、hsa-miR-483-3p和hsa-miR-1-3p可能作为孤独症辅助诊断的生物标记物，该miRNA通过调控孤独症风险基因，可能参与孤独症的发病。