Table1.DOCX

Enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) remain the predominant etiological agents of hand, foot, and mouth disease (HFMD). The clinical manifestations caused by the two viruses are obviously different. CV-A16 usually triggers a repeated infection, and airway epithelial integrity is often the potential causative factor of respiratory repeated infections. Our previous studies have demonstrated that there were some differentially expressed miRNAs involved in the regulation of adhesion function of epithelial barrier in EV-A71 and CV-A16 infections. In this study, we compared the differences between EV-A71 and CV-A16 infections on the airway epithelial barrier function in human bronchial epithelial (16HBE) cells and further screened the key miRNA which leaded to the formation of these differences. Our results showed that more rapid proliferation, more serious destruction of 16HBE cells permeability, more apoptosis and disruption of intercellular adhesion-associated molecules were found in CV-A16 infection as compared to EV-A71 infection. Furthermore, we also identified that microRNA-4516 (miR-4516), which presented down-regulation in EV-A71 infection and up-regulation in CV-A16 infection was an important regulator of intercellular junctions by targeting Poliovirus receptor related protein 1(PVRL1). The expressions of PVRL1, claudin4, ZO-1 and E-cadherin in CV-A16-infected cells were significantly less than those in EV-A71-infected cells, while the expressions of these proteins were subverted when pre-treated with miR-4516-overexpression plasmid in EV-A71 infected and miR-4516-knockdown plasmid in CV-A16 infected 16HBE cells. Thus, these data suggested that the opposite expression of miR-4516 in EV-A71 and CV-A16 infections might be the initial steps leading to different epithelial impairments of 16HBE cells by destroying intercellular adhesion, which finally resulted in different outcomes of EV-A71 and CV-A16 infections.

肠道病毒71型（Enterovirus 71, EV-A71）与柯萨奇病毒A16型（coxsackievirus A16, CV-A16）仍是手足口病（hand, foot, and mouth disease, HFMD）的主要致病病原体。两种病毒引发的临床表现存在显著差异：CV-A16通常会导致重复感染，而气道上皮完整性往往是呼吸道重复感染的潜在致病因素。本课题组既往研究证实，在EV-A71与CV-A16感染过程中，存在若干参与调控上皮屏障黏附功能的差异表达微小RNA（microRNA, miRNA）。本研究对比了EV-A71与CV-A16感染对人类支气管上皮（16HBE）细胞气道上皮屏障功能的影响差异，并进一步筛选出介导此类差异形成的关键微小RNA。结果显示，相较于EV-A71感染，CV-A16感染的16HBE细胞增殖速度更快、细胞通透性破坏程度更严重、细胞凋亡水平更高，且细胞间黏附相关分子的紊乱程度更显著。此外，本研究还证实，微小RNA-4516（microRNA-4516, miR-4516）可通过靶向结合脊髓灰质炎病毒受体相关蛋白1（Poliovirus receptor related protein 1, PVRL1）调控细胞间连接，该miRNA在EV-A71感染中呈下调表达，而在CV-A16感染中则呈上调表达。CV-A16感染细胞中PVRL1、闭合蛋白4（claudin4）、紧密连接蛋白ZO-1及E-钙粘蛋白的表达水平均显著低于EV-A71感染细胞；而在EV-A71感染的16HBE细胞中预转染miR-4516过表达质粒，或在CV-A16感染的16HBE细胞中预转染miR-4516敲低质粒，均可逆转上述蛋白的表达差异。综上，本研究数据表明，EV-A71与CV-A16感染中miR-4516的相反表达模式，可能是通过破坏细胞间黏附进而引发16HBE细胞上皮损伤差异的起始环节，最终导致两种病毒感染出现不同的临床结局。