miR-1303靶向调控MMP9介导EV71和CA16感染中血脑屏障通透性的改变

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16), as major etiological pathogens of hand, foot and mouth disease (HFMD), caused severe neurological complications which have been emerged as a serious threat to the public health among infants and young children. However, the underlying mechanisms which make the two viruses entry into central nervous system (CNS) remain elusive. Our previous studies have demonstrated that matrix metalloproteinase 9 (MMP9) presented significantly increased in rhesus monkey and human umbilical vein endothelial cells (HUVECs) infected with EV71 and CA16. Moreover, we also found that MMP9 might be the direct target gene of miR-1303 by analysis of high-throughput sequencing. Herein, based on the above research, we aim to adopt an in vitro model using HUVECs and an in vivo model using scavenger receptor class B member 2 (SCARB2) humanized mouse to investigate the regulation mechanisms between viruses and miR-1303, and further explore whether miR-1303 direct targets MMP9 which might degrade tight junctions or adherens junctions molecules and ultimately facilitate EV71 and CA16 to destruct blood-brain barrier and invade CNS. Additionally, clinical samples were used to further verify the correlations between the expression levels of miR-1303 and MMP9 and the severe degree of EV71- and CA16-infected patients. These findings might not only offer novel targets for clinical therapy, but also provide an early warning molecular for the diagnosis of severe HFMD cases.

EV71和CA16是手足口病最重要的病原体，二者能够引起中枢神经系统损伤，对婴幼儿的健康构成巨大的威胁，但二者进入中枢神经系统的机理仍不清楚。我们前期研究发现MMP9在EV71和CA16感染的恒河猴体内及人血管内皮细胞上都显著性上升，且microRNA测序分析结果提示miR-1303可能在EV71和CA16感染中靶向调控MMP9。本课题拟在前期研究的基础上，利用体外细胞和体内SCARB2人源化小鼠模型，探讨病毒调控miR-1303的方式，以及miR-1303是否能靶向调控MMP9介导的血管内皮细胞之间的连接分子的降解过程，最终阐明EV71和CA16打破血脑屏障进入中枢神经系统的机制，在此基础上结合临床样本分析，寻找miR-1303、MMP9与EV71和CA16感染患者临床症状严重程度的相关性，以期为EV71和CA16感染的临床治疗提供候选靶标以及为重症手足口病患者的诊断提供早期预警分子。

EV71和CA16是手足口病最重要的病原体，二者能够引起中枢神经系统损伤，对婴幼儿的健康构成巨大的威胁，但二者进入中枢神经系统的机理仍不清楚。我们发现MMP9在EV71和CA16感染的恒河猴体内及人血管内皮细胞上都显著性上升，此外，我们对EV71和CA16感染后的人脐静脉血管内皮细胞（HUVECs）的microRNA的表达谱进行了分析，结果提示miR-1303可能在EV71和CA16感染中靶向调控MMP9。我们在体外的血脑屏障(BBB)细胞模型上证实了miR-1303确实可以靶向作用于MMP9，进一步研究证实MMP9的可以降解细胞与细胞之间的紧密连接（TJ）和黏附连接分子（AJ）从而改变BBB的通透性。这些现象也在CA16的恒河猴婴猴感染模型中得到了验证，给予感染CA16的婴猴同时注射MMP9重组蛋白会导致其与仅仅感染CA16的婴猴相比脑部的连接分子的破坏更加明显，同时更多的病毒穿透BBB进入了脑部，且肺部和丘脑的病毒损伤更加严重；而给予注射TIMP-1（MMP9抑制剂）则能显著缓解CA16感染的恒河猴婴猴脑部连接分子的降解，阻止更多的病毒进入脑部和改善肺部和丘脑的病理损伤。以上的结果基本能表明了：EV71/CA16感染宿主后能通过抑制miR-1303的表达从而促进MMP9的释放，释放的MMP9则能够降解细胞与细胞之间的连接，从而使BBB的通透性增加，这样导致更多的病毒、炎性因子和炎性细胞浸润，最终导致中枢神经系统（CNS）损伤。那么MMP9除了介导EV71/CA16进入CNS外，是否会对已经存在于CNS中的病毒存在影响？我们选择了神经系统中的免疫细胞-小胶质细胞作为研究对象，我们发现MMP9的表达会促进EV71在小胶质细胞中的复制，但是MMP9并不会抑制小胶质细胞中干扰素（IFN）的表达，进一步研究发现MMP9会结合并降解干扰素受体（IFNAR），从而使得IFN/JAK/STAT/ISGs通路收到抑制，最终促进EV71的复制，此外MMP9还会促进NLRP3介导的细胞焦亡反应导致炎性因子IL-1β的释放，进一步加剧CNS的损伤。本研究为EV71和CA16感染的临床治疗提供了候选靶标以及为重症手足口病患者的诊断提供了早期预警分子。