长链非编码RNA-RP11-54O7.17下调干扰素刺激基因15（ISG15）表达促进 EV71感染致重症的机制研究

Since 2008, there have been more than a million HFMD cases reported in China every year, with thousands of severe cases. Previous studies have shown that more than 80% of the HFMD severe cases in China are caused by enterovirus 71 (EV71), however the molecular mechanism remains poorly understood. We have performed a high throughput sequencing of lncRNA, microRNA, and mRNA for blood samples from mild and severe HFMD patients with EV71 infection, and identified a differential expression network of lncRNA RP11-54O7.17, miR-130b-5p and ISG15 related to the severity of HFMD. In this study, we will further investigate the correlation of their expression changes in severe HFMD cases. Lentiviral vector and RNA interference approaches will be used to construct the RP11-54O7.17-overexpressed/silenced cell lines and transgenic mice to study the effect of RP11-54O7.17 on regulating the expression of ISG15 in the initiation and progression of severe HFMD cases in vivo and in vitro. In addition, the luciferase signal system and immunoprecipitation will be used to explore the mechanism of RP11-54O7.17 in regulating the expression of ISG15 via either targeted regulating or acting as the molecular sponge of miR-130b-5p. Accordingly, we will further elucidate the molecular mechanism of development and progression of severe HFMD.

2008年以来我国每年报告上百万手足口病（HFMD）病例，数千例重症病例，其中80%以上的HFMD重症由肠道病毒71型（EV71）引起，但发生机制并不清楚。课题组前期通过对EV71感染导致的HFMD轻症和重症患者外周血进行高通量测序，筛选出一个与HFMD严重程度相关且差异表达的lncRNA RP11-54O7.17、miR-130b-5p及ISG15相互作用网络。本项目拟在此基础上进一步验证三种不同性质RNA在HFMD重症患者中的表达关系，并用慢病毒载体、RNA干扰技术构建RP11-54O7.17过表达/沉默细胞系及转基因小鼠，在细胞和动物水平研究其调控ISG15表达对HFMD重症发生、发展的作用，通过荧光素酶信号系统及免疫共沉淀等技术探讨RP11-54O7.17靶向调控或作为miR-130b-5p的“分子海绵”调控ISG15表达的机理，进一步阐明HFMD重症发生、发展的分子机制。

2008年以来我国每年报告上百万手足口病（HFMD）病例，数千例重症病例，聚集性暴发次数显著增加，其中80%以上的HFMD重症由肠道病毒71型（EV71）引起，对EV71引起HFMD重症发生、发展的机制研究是当前科技前沿的热点和难点问题，目前对这一机制尚不清楚。随着近些年转录组测序技术的广泛应用，现已有成千上万的长链非编码RNA（lncRNA）相继从单细胞真核生物到人类被鉴定出来，大量研究表明lncRNA在表观遗传学调控中扮演了越来越重要的角色，以lncRNA为切入点，开展对疾病发生机制的研究成为基础研究的新兴领域。. 课题组前期通过对EV71感染导致的HFMD轻症和重症患者外周血进行高通量测序，筛选出一个与HFMD严重程度相关且差异表达的lncRNA RP11-54O7.17、miR-130b-5p及ISG15相互作用网络。本项目在此基础上通过扩大样本量验证了三种不同性质RNA在HFMD重症患者中的表达关系，证实了EV71感染诱导IncRNA RP11-5407.17表达水平下调。利用腺相关病毒载体、RNAi腺相关病毒载体构建RP11-54O7.17过表达/沉默细胞系，定量EV71 VP1蛋白和宿主ISG15蛋白转录和表达水平的动态变化，发现RP11-54O7.17高表达能够影响宿主ISG15的复制效率，上调ISG15表达，实现对EV71 VP1蛋白表达的负调控，证实了RP11-54O7.17在抑制EV71复制及其感染性中的重要作用。本项目明确了RP11-54O7.17差异表达调控ISG15对HFMD重症发生、发展的作用，初步讨论了RP11-54O7.1靶向调控或作为miR-130b-5p的“分子海绵”调控ISG15表达的作用途径，进一步阐明了HFMD重症发生、发展的分子机制，为后续开发针对HFMD诊疗的特异性和靶向性RNA制剂提供理论依据。