Investigations On Different MicroRNA Alterations That Contribute To Diverse Pathogenic Mechanisms Following EV71 And CA16 Infections

Hand,foot,and mouth disease(HFMD)frequently occurs in infants and children below 5 years of age.Enterovirus 71(EV71)and Coxsackievirus A16(CA16)and are the predominant etiological agents of HFMD.Although they both belong to the human enterovirus A(HEV-A)species of the Picomaviridae family,different clinical manifestations are caused by infections with these viruses.microRNAs(miRNAs),encoded single-stranded RNAs of approximately 22 nucleotides in length,act as key regulators of gene expression at the post-transcriptional level by complementary pairing with the 3'-untranslational region(3'-UTR)of messenger RNAs(mRNAs)for translational repression or degradation.Moreover,miRNAs participate in various physiological and pathological processes.Currently,it has reported that miRNAs also play an important role in infectious diseases.Therefore,this study aimed to explore the differential expressed miRNAs induced by EV71 and CA16 infections with miRNA high throughput sequencing(HTS)technique,and to further investigate the roles of these differential expressed miRNAs in EV71 and CA16 infections.These results might provide new clues to explain the differences in clinical symptoms and pathological mechanisms in EV71 and CA16 infections.In the current study,we used EV71 and CA16 to infect 16HBE cells and then adopted miRNA HTS analysis.The results showed that 201 miRNAs exhibited remarkable differences in expression.Of these,65 miRNAs,including 58 known and 7 novel miRNAs,presented opposite trends in EV71-and CA16-infected samples.Subsequently,we mainly focused on the 56 known differentially expressed miRNAs by further screening for targets prediction.GO and pathway analysis of these targets demonstrated that 18 biological processes,7 molecular functions,1 cellular component and 123 pathways were enriched.Among these pathways,Cadherin signaling pathway,Wnt signaling pathway and angiogenesis showed significant alterations.The regulatory networks of these miRNAs with predicted targets,GOs,pathways and transcription factors were determined,which suggested that miRNAs displayed intricate regulatory mechanisms during the infection phase.Consequently,we specifically analyzed the hierarchical GO categories of the predicted targets involved in adhesion.These results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to airway epithelial barrier function.Subsequently,we continued to compare the differences between EV71 and CA16 infections in 16HBE cells.The results showed that more rapid proliferation,apoptosis and disruption of cell-cell contact were observed in CA16 infection compared to EV71 infection.Furthermore,CA16 caused more serious destruction of 16HBE cells permeability relative to EV71.However,the levels of type I-interferon(IFN-I)production-associated molecules and chemokines induced by CA16 infection were lower than those produced by EV71 infection.Additionally,we identified miR-4516,which presented down-regulation in EV71 infection and up-regulation in CA16 infection,as an important regulator of intercellular junctions by targeting PVRL1.Moreover,in CA16-infected cells,PVRL1,claudin4,ZO-1 and E-cadherin were significantly reducted in location and protein expression compared with those in EV71-infected cells,while the above phenomena were subverted with miRNA-4516-up pre-treatment in EV71 infection and miRN A-4516-down pre-treatment in CA16 infection.These data suggested that that the opposite expression trends of miR-4516 in EV71 and CA16 infections might have a crucial role in the different degrees of impairment of 16HBE cells by destroying cell-cell contacts,which provide the novel viewpoint that these different degrees of impairment of the 16HBE cells may be the initial steps that lead to different outcomes of EV71 and CA16 infections.Next,we utilized EV71 and CA16 to infect the PBMCs of rhesus monkey and also performed miRNA HTS analysis.The results showed that 106 known and 13 novel miRNAs exhibited significantly differences and 32 key miRNAs among them for target prediction presented opposite trends in the EV71-and CA16-infected samples.GO and pathway analysis of the predicted targets enriched 14 biological processes,10 molecular functions,8 cellular components and 104 pathways.Subsequently,regulatory networks of miRNA-transcription factors,miRNA-predicted targets,miRNA-GOs and miRNA-pathways were constructed to reveal the complex regulatory mechanisms of miRNAs during the infection phase.Ultimately,we analyzed hierarchical GO categories of the predicted targets involved in immune system process,which indicated that the innate and adaptive immunity following EV71 and CA16 infection may be remarkably distinct.Our findings could provide a valuable basis for further studies on the regulatory roles of miRNAs related to the different immune responses caused by EV71 and CA16 infections.Finally,we carried out the comparative analysis on the differences in CD1c+DC of rhesus monkey subjected to EV71 and CA16 infections.The results displayed that in EV71 infection,the expression of IFN-? were significantly decreased,virus copies,PFUs of virus and the number of virus infection were increased,and the expressions of the transcription factors and cytokines related to Th2 and Tfh cells were markedly increased;while in CA16 infection,the expression of IFN-I were remarkably up-regulated,virus copies,PFUs of virus and the number of virus infection were down-regulated,and the expressions of the transcription factors and cytokines related to Thl and Treg cells were notably up-regulated.These findings concluded that the different innate and adaptive immunity caused by EV71 and CA16 infections in CD1c+DC might be tightly associated with the distinct clinical symptoms and pathogenesis of HFMD.