MiR-124/STAT3通路影响EV71感染和复制的分子机制

Enterovirus 71 (EV71) has been the main causative agent of hand, foot, and mouth disease outbreaks in recent years. The virus can also cause children severe neural diseases, and even death. The inflammatory cytokine storm and cellular apoptosis were reportedly associated with the severe consequences..Many publications showed that the proinflammatory mediators (such as COX2, VCAM-1, and TNF-α) were induced upon EV71 infection by activated some signaling pathways, such as C-Src (sarcoma kinase), EGFR (epidermal growth factor receptor, or simplified as ErbB-1), PDGFR (Platelet-derived growth factor receptor), NF-κB, and MAPK signal cascades. C-Src, EGF, and PDGF are activators of STAT3 signaling pathway. NF-κB and MAPK could also interact with STAT3. Furthermore, STAT3 has been reported with the activity of anti-apoptosis. However, the role of STAT3 in the replication of EV71 is unknown. We have found that high levels of phosphorylation of STAT3 inhibited EV71 infection in RD cells, but overexpression of miR-124 promoted the viral replication. Given the fact that miR-124 can modulate STAT3 and its downstream signaling pathways by directly targeting STAT3, or indirectly by targeting the upstream genes of STAT3 signaling pathways, such as IL-6R (a component of IL-6 receptor complex), it was deduced that miR-124 could be involved in EV71 infection via targeting STAT-3. Consequently, this proposal will focus on the molecular mechanisms of miR-124/STAT3 pathway underlying the viral replication. By the way of loss- and gain-of-function of genes, we will identify the roles of miR-124/STAT3 pathway in the viral infection, and determine whether miR-124 promoting the viral replication is through targeting STAT3, or inhibiting its phosphorylation, and further ananlyze which downstream genes of STAT3 were ascribed to the antiviral effects induced by STAT3. Simultanously, we will identify which viral protein or functional genectic region of EV71 induced the responses of host miR-124 and STAT3. The results would be beneficial to understand the interactions between the host and the virus, and promote further researches for the new antivirals.

EV71感染除引起婴幼儿手足口病外，还容易引起神经源性重症，甚至死亡。而诱导大量致炎症因子，形成细胞因子风暴和细胞凋亡是导致重症的重要原因。.EV71诱生致炎症因子及其依赖的信号通路很多与STAT3相关，STAT3还能抗细胞凋亡。我们前期研究也发现STAT3可抑制EV71感染，而miR-124下调STAT-3及其磷酸化，并促进EV71复制。因此推测miR-124和STAT3可能参与EV71感染的调控，并形成上下游关系。然而它们在EV71感染时究竟有何作用，是否形成上下游关系，miR-124/STAT3通路又调控哪些基因影响EV71感染，以及EV71用何种病毒基因或蛋白影响miR-124/STAT3通路？上述问题并不清楚，因此，本课题拟利用基因沉默、过表达等技术，试图阐明miR-124/STAT3通路与EV71如何相互作用，以加深对EV71复制过程的理解，并为研制抗EV71药物提供新思路。

新型肠道病毒71型（Enterovirus type 71, EV71）感染常导致婴幼儿手足口病（Hand, foot, and mouth disease, HFMD），但也有部分患者并发神经源性重症，甚至死亡。EV71致病机制主要与病毒逃逸宿主免疫、诱发炎症因子风暴，以及导致细胞死亡有关。STAT3在宿主免疫应答、诱导炎症因子产生、以及调控细胞凋亡过程中起着十分重要作用。基于我们前期的初步发现，STAT3以及调控其表达的miR-124分子，对EV71的感染有明显影响的结果，本项目进一步利用了多种EV71感染细胞模型，采用基因过表达、Knock-down或Knock-out技术，系统地研究了STAT3和miR-124对EV71感染的作用。结果证实了STAT3的表达和磷酸化激活可抑制EV71的感染，而EV71在感染过程中，可通过上调miR-124的表达，并利用miR-124下调STAT3 mRNA水平，起着拮抗STAT3的抗病毒作用，促进病毒的感染和复制。此外，由于IL-6R可与IL-6结合并激活STAT3信号通路，且IL-6R也是miR-124的靶基因，因此在本项目的支持下，我们拓展研究了IL-6R对EV71感染的影响，发现与STAT3类似，IL-6R也可抑制EV71的感染，并且都受miR-124的调控。对EV71全基因组扫描分析发现，多个病毒基因可能与宿主细胞STAT3的磷酸化激活有关；而通过STAT3 Knock-down与病毒感染前后细胞转录组的比较，结合转录因子与启动子相互作用分析，初步发现JUN和EGR1等基因可能在STAT3影响EV71感染过程中起一定作用。上述研究结果表明，控制miR-124及其下游靶基因IL-6R和STAT3的表达和激活对EV71感染的治疗可能具有重要作用，这对深入了解EV71感染和复制过程，发现新的抗病毒药物具有一定的提示意义。