血凝性脑脊髓炎病毒致脑损伤相关microRNA的鉴定与功能分析

Porcine hemagglutinating encephalomyelitis virus (PHEV) mainly invades the central nervous system of host, and the brain injury with encephalomyelitis caused by PHEV is an important reason for acute death of infected host, yet its pathogenesis remains poorly understood. In recent researches, some evidences had shown that cellular miRNAs have key regulatory roles between virus and host in many viral nervous systemic diseases. However, little is known if there are any miRNAs involving in regulating mRNA expression of host in the process of PHEV infection. To gain more insight into the pathogenesis of PHEV, and clarify the host miRNAs function in the proceeding of brain damage caused by PHEV infection, differentially expressed patterns of cellular miRNAs correlated with brain tissues damage will be selected by using miRNA microarray profiles, which from the cerebral cortex of mice infected with PHEV in this study. The miRNA molecules correlated with PHEV infection and its target genes will be predicted by bioinformatics assays. Then the differentially expressed miRNAs will be further identified in the primary neuron cells and Balb/c mice by gene silencing and over-expression assays, and its functional analysis also will be performed. The prospective results will not only help us to understand deeply the regulation mechanism on cellular miRNA correlated with PHEV infection, but also provide significant fundamental basis for analyzing the molecular mechanism of PHEV invasion.

猪血凝性脑脊髓炎病毒（PHEV）主要侵害中枢神经系统，所引起的脑组织损伤是导致感染宿主急性死亡的重要原因。对一些病毒性神经系统疾病的研究发现，病毒侵染会引起宿主细胞microRNA(miRNA)表达出现上调或下调，从而证实宿主miRNA表达谱的变化与病毒侵染存在密切关系。是否有宿主miRNA通过调控宿主基因的表达而参与PHEV的感染和致病过程，尚有待于研究。本项目在前期建立PHEV稳定感染小鼠模型基础上，利用miRNA芯片对PHEV感染前后小鼠大脑皮质miRNA表达谱进行研究，筛选差异表达显著的miRNA，在生物信息学分析基础上，鉴定与PHEV致病相关的miRNA及其靶基因；应用基因沉默和过表达等技术从体内外两个角度进一步对目标miRNA进行鉴定和功能分析。该项研究不仅有助于阐明PHEV感染致病过程中由宿主miRNA介导的调控机制，而且将为深入解析PHEV的分子致病机制提供重要理论依据。

本研究主要探究PHEV感染过程中是否有宿主miRNA通过调控宿主基因的表达而参与PHEV的感染和致病过程。在建立PHEV稳定感染小鼠模型基础上，利用基因芯片技术对PHEV感染前后小鼠大脑皮质miRNA表达谱和mRNA表达谱进行检测，筛选得到差异miRNAs和差异基因并对其验证。应用生物信息学方法建立差异基因和差异miRNA的调控网络，在调控网络中发现mmu-let-7b、miR-142-5p和miR-21a-5p在病毒致病过程中起着重要的调控作用。其中，mmu-let-7b通过调控已知靶基因Pro-Caspase-3在PHEV诱导神经凋亡过程中起重要作用。利用生物信息学软件分析目的miRNA分子的潜在靶基因，选择与病毒感染、神经功能紊乱等相关信号通路的调控相关性较大的基因Ulk1（Unc-51-like kinase 1）和Caskin1（CASK interacting protein 1），分别作为miR-142-5p和miR-21a-5p的候选靶基因。经双荧光素酶报告基因系统、western blotting、real-time PCR等方法验证,确定UIK1和Caskin1分别为miR-142-5p和miR-21a-5p的靶基因。在小鼠原代神经元及小鼠神经瘤母细胞(N2a细胞)中分别过表达或抑制miR-142-5p或Ulk1表达的研究证实,在PHEV致神经损伤过程中,miR-142-5p通过抑制UIk1的表达进而抑制神经元轴突的延伸，干扰病毒的复制。在N2a细胞上分别有效的过表达或者抑制miR-21a-5p或Caskin1的表达后接种病毒，发现其可促进病毒的复制。在BALB/c小鼠脑内注射一定量的miR-142-5p antagomirs或miR-21a-5p antagomirs后，滴鼻接种PHEV，发现antagomirs可减缓miRNA对靶基因 mRNA的抑制，小鼠脑组织中PHEV感染量也有所降低，且感染小鼠发病延迟且存亡率明显增高。本研究不仅有助于阐明PHEV感染致病过程中由宿主miRNA介导的调控机制，而且为深入解析PHEV的分子致病机制提供重要理论依据。