基于lncRNA R-AS1调控天然免疫应答抵抗重症肠道病毒EV71感染机制的研究

EV71 is an important kind of infectious virus, and some EV71 strains can cause severe clinical complications. After the virus infects the host, the virus RNA is recognized by the host pathogen recognition receptors, activates the cellular antiviral innate immune response, and suppresses the virus replication. lncRNA (long noncoding RNA, lncRNA) is a long non encoding RNA molecules widely existed in organisms, it has been reported lncRNA has the immune regulatory function in the cellular process.To further clarify the molecular mechanism of lncRNA regulation of the innate immune response in the process, this study will use RNA-seq to find related regulation of host innate immunity antiviral lncRNA as the pointcut, by means of bioinformatics analysis and molecular biology methods, to confirm the non encoding characteristics and sequence of lncRNA; Through analyzing the relevance of lncRNA expression spectrum with mRNA to search the interaction of lncRNA with interferon stimulated gene (ISGs); The lentiviral integration,RNA interference or CRISPR methods will be used to establish the over expression of lncRNA and gene knockout cell lines, to in vitro validate of lncRNA and target ISGs interaction; The methylation inhibition/activation and chromatin immunoprecipitation methods will be used to identify the interaction mechanism between lncRNA and ISGs. lncRNA mutants were established and transfected into cell to search for the functional sequence of lncRNA. The research results will better illustrate the mechanism and function of lncRNA effect on the control of host innate immune response against EV71.

肠道病毒（EV71）是一种重要的传染性病毒，有些EV71能引起临床并发症的重症。病毒感染宿主后，病毒RNA被宿主的病原识别受体所识别，激活细胞抗病毒天然免疫应答，抑制病毒复制。lncRNA(long noncoding RNA, lncRNA)是广泛存在于生物体内的长链非编码RNA分子，已有报道证明lncRNA在细胞生命过程中具有免疫调节功能，为进一步阐明lncRNA调控天然免疫应答过程中的分子机制，本研究拟采用RNA-seq测序技术寻找与调控宿主天然免疫抗病毒有关的lncRNA为切入点，借助生物信息学分析和分子生物学手段，确认目标lncRNA的非编码特征和全长序列；进行lncRNA与mRNA表达谱变化关联分析，筛选与lncRNA相互作用的干扰素刺激基因(ISGs)；通过慢病毒、siRNA或CRISPR技术建立lncRNA过表达和敲除细胞系，细胞水平验证目标lncRNA与ISGs相互作用；采用甲基化抑制/激活和染色质免疫共沉淀技术探索两者相互作用机理；建立并转染lncRNA突变体以寻找其功能序列。通过以上研究，旨在阐明lncRNA对调控宿主抗EV71天然免疫应答中作用机理和功能的影响。

手足口病是由肠道病毒感染引起的儿童常见传染病，其发病率与死亡率高居我国丙类传染病之首。目前，肠道病毒EV71型（EV71）已被确定为国内引起手足口病的主要流行病毒，当病毒侵染宿主后，其核酸或蛋白质组分可被宿主病原识别受体（PRR）鉴别出来，进而激活一系列转录因子，启动干扰素刺激基因（ISGs）表达激活天然免疫应答抵抗病毒感染。长链非编码RNA（lncRNA）是一类转录本大于200 nt的RNA分子，绝大多数不能编码蛋白质，因其能参与转录及转录后调控，与肿瘤转移，抗病毒天然免疫等密切相关，因此，研究lncRNA在宿主抗肠道病毒天然免疫中扮演重要的角色具有重要意义。.本研究利用RNA-Seq和生物信息学分析技术成功筛选到与EV71复制相关的lncRNA R-AS1，EV71感染诱导lncRNA R-AS1表达水平下调。通过体外实验验证lncRNA R-AS1过表达可显著提高EV71的复制效率，病毒滴度显著增加。体内实验证明lncRNA R-AS1高表达可增强EV71对乳鼠的致病力，主要的靶器官肺脏和后肢肌病变严重。lncRNA R-AS1下调可降低病毒复制水平，炎症反应减轻，提高感染乳鼠的存活率。转录组测序发现lncRNA R-AS1过表达后，有1060种差异表达基因，Go和KEGG分析确定多种参与天然免疫途径的ISGs基因。进一步通过RNA pull-down和质谱分析发现与lncRNA R-AS1相互作用的分子为主要穹隆蛋白（MVP），lncRNA R-AS1定位在细胞质，通过序列205-397形成的颈环臂与MVP相互作用负调控EV71的复制，因此该颈环臂是lncRNA R-AS1发挥功能的主要结构域。.EV71感染后可特异性诱导lncRNA R-AS1下调，提示lncRNA R-AS1可作为手足口病诊断的一个潜在分子标志物。本研究揭示了lncRNA R-AS1作为负调控因子与MVP结合参与病毒复制过程。因此，lncRNA R-AS1的发现和功能鉴定为将来设计新的抗病毒策略奠定了良好的基础，也为手足口病的诊断和治疗提供了新思路。