miR-1303 regulates BBB permeability and promotes CNS lesions following CA16 infections by directly targeting MMP9

Coxsackievirus A16 (CA16) is a member of the Picornaviridae family and causes mild and self-limiting hand, foot, and mouth disease (HFMD) in infants and young children. CA16 infection can also progress to central nervous system (CNS) complications; however, the underlying mechanism by which CA16 penetrates the blood-brain barrier (BBB) and then causes CNS damage remains unclear. This study aimed to explore the mechanism of CA16 neurotropic tropism by establishing an in vitro BBB model with CA16 infection and an in vivo CA16 rhesus monkey infant infection model. The results showed that CA16 infection induced increased permeability of the BBB accompanied by upregulation of matrix metalloproteinase 9 (MMP9) expression. Subsequently, high-throughput miRNA sequencing technology and bioinformatics analysis revealed that miR-1303 may regulate BBB permeability by targeting MMP9. Next, we used dual-luciferase, qRT-PCR, and western blot assays to provide evidence of MMP9 targeting by miR-1303. Further experiments revealed that CA16 infection promoted the degradation of junctional complexes (Claudin4, Claudin5, VE-Cadherin, and ZO-1), likely by downregulating miR-1303 and upregulating MMP9. Finally, EGFP-CA16 infection could enter the CNS by facilitating the degradation of junctional complexes, eventually causing neuroinflammation and injury to the CNS, which was confirmed using the in vivo rhesus monkey model. Our results indicate that CA16 might penetrate the BBB and then enter the CNS by downregulating miR-1303, which disrupts junctional complexes by directly regulating MMP9 and ultimately causing pathological CNS changes. These results provide new therapeutic targets in HFMD patients following CA16 infection.

柯萨奇病毒A16（Coxsackievirus A16, CA16）隶属于小RNA病毒科（Picornaviridae），可导致婴幼儿罹患轻症、自限性的手足口病（hand, foot, and mouth disease, HFMD）。CA16感染还可能进展为中枢神经系统（central nervous system, CNS）并发症，但目前CA16穿透血脑屏障（blood-brain barrier, BBB）并进而造成中枢神经系统损伤的潜在分子机制仍未阐明。本研究旨在通过构建CA16感染的体外血脑屏障模型以及体内恒河猴幼崽感染模型，探究CA16的神经嗜性机制。研究结果显示，CA16感染可升高血脑屏障通透性，并伴随基质金属蛋白酶9（matrix metalloproteinase 9, MMP9）表达上调。随后，通过高通量miRNA测序技术与生物信息学分析发现，miR-1303可能通过靶向调控MMP9来影响血脑屏障通透性。后续我们采用双荧光素酶实验、实时荧光定量PCR（qRT-PCR）以及蛋白质免疫印迹（western blot）实验，验证了miR-1303对MMP9的靶向调控作用。进一步实验表明，CA16感染可能通过下调miR-1303并上调MMP9，促进紧密连接蛋白4（Claudin4）、紧密连接蛋白5（Claudin5）、血管内皮钙粘蛋白（VE-Cadherin）以及闭锁小带蛋白1（ZO-1）组成的紧密连接复合体的降解。最后，通过体内恒河猴模型验证发现，增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）标记的CA16可通过促进紧密连接复合体降解侵入中枢神经系统，最终引发神经炎症与中枢神经系统损伤。本研究结果表明，CA16可能通过下调miR-1303，直接调控MMP9以破坏紧密连接复合体，进而穿透血脑屏障侵入中枢神经系统，最终引发中枢神经系统病理性改变。上述发现为CA16感染所致手足口病患者提供了全新的治疗靶点。