The Expression Profile And Preliminary Function Study Of Long Non-conding RNAs In Coxsackie Virus B5 Infected Human Neuroblastoma Cells

Coxsackie virus B5(CVB5)is one of the main pathogens that cause hand,foot and mouth disease(HFMD).It mainly infects infants under 5 years and mostly in summer and autumn.CVB5 are spread by the fecal-oral route,where it is proliferated in the intestine and then spread to other tissues and organs,causing other diseases.Clinical symptoms typically manifest as fever,mucosal rash and herpes.It can resultsin neurological complications and even death in severe cases.In resent years,HFMD caused by CVB5 has been epidemic trend and posed a serious threat to infants.However,there is no treatment method effective vaccine for CVB5 infected HFMD and neurological diseases at present.Long noncoding RNA(lncRNA)are molecules of RNA with a length greater than 200 nucleotides,they have multiple biological functions,such as guide,sponges,decoys and interacts with proteins.At the same time,more and more studies have proved that t here is a close relationship between lncRNA and virus-host.Virus infection can cause the differential expression of host and virus-encoded lncRNAs.They play a role by regulating immune signal pathways,viral genome transcription and translation,and host cell me Htabolites.However,the regulatory mechanism of most lncRNAs after virus infection is still unknown.Therefore,this study established a cell model of CVB5 infection of human rhabdomyosarcoma cells(RD)and human neuroblastoma cells(SHSY5Y)We selected two significantly different lncRNAs to conduct a preliminary exploration on the role of regulating viral replication,in order to clarify the mechanism of lncRNA in CVB5 replication,so as to lay the foundation for the diagnosis,prevention and treatment of viral diseases.In this study,we first explore the optimal infection conditions of CVB5,and developed the CVB5 infection model.After CVB5 infection with neuronal cells,SHSY5 Y cells were confirmed to be the most suitable human nerve cell by observing cytologic changes,detecting CVB5 VP1 protein expression and cell viability identification.Meanwhile,after 1MOI CVB5 infects RD and SHSY5 Y cells,it is found that the optimal infection time for CVB5 to infect RD and SHSY5 Y cells is 24 h by measuring TCID50 and cell viability.What’s more,we analyzed and identified the lncRNA obtained after CVB5 infection of RD and SHSY5 Y cells through RNA-seq sequencing technology.The results showed that 508 lncRNAs were up-regulated and760 lncRNAs were down-regulated in infected RD cells.The proportions of linc RNA,sense-intronic,antisense,and sense-overlopapping were 46.2%,1.0%,28.6%,and24.1%,respectively;In SHSY5 Y cells,792 lncRNAs were up-regulated and 811 lncRNAs were down-regulated.The proportions of linc RNA,sense-intronic,antisense,and sense-overlopapping were 46.2%,1.0%,28.6%,and 24.1%,respectively.Next,through the analysis of lncRNA GO enrichment and KEGG function,it was found that the differential lncRNAs of RD cells infected were mainly related to processes such as endoplasmic reticulum protein processing,lupus erythematosus and viral myocarditis.The differential lncRNAs of SHSY5 Y cells were mainly related to neurotrophic signaling pathway and erb B signaling pathway.At the same time,we selected 8 lncRNAs with significant differences for qRT-PCR verification,and the results were consistent with the sequencing data,so that subsequent experimental studies could be carried out.Finally,we screened out lncRNA-SMIM18 and lncRNA-IL12 A that are differentially expressed only in SHSY5 Y cells for research,in order to explore the effect of lncRNA on the replication of CVB5 among the lncRNAs verified by qRT-PCR.We constructed lncRNA-SMIM18 and lncRNA-IL12 A silencing vectors.After transfecting the cells,we found that CVB5 VP1 protein expression and virus titer increased significantly.The results showed that both lncRNAs inhibited virus replication.Subsequently,KEGG analysis showed that lncRNA-SMIM18 was enriched in the JAK-STAT signal pathway,while lncRNA-IL12 A was enriched in the Wnt pathway signal,which indicated that the two lncRNAs could regulate virus replication through different mechanisms.Next,we analyzed the regulatory effects of two lncRNAs on key genes in the pathway by qRT-PCR.The results showed that lncRNA-SMIM18 positively regulated the gene expression of IFTM2,OASL and ISG20,and lncRNA-IL12 A negatively regulates the expression of related moleculesβ-catenin and target genes C-myc and Cyclin D1 in the Wnt signaling pathway.In summary,this study established a model of CVB5 infection of RD and SHSY5 Y cells,and analyzed the lncRNA after virus infection of host cells through RNA-seq technology,and selected lncRNA-SMIM18 and lncRNA-IL12 A as the research objects.It is clear that lncRNA-SMIM18 regulates CVB5 replication by regulating the JAK-STAT pathway,and lncRNA-IL12 A regulates the Wnt signaling pathway to inhibit viral replication.This topic explores the regulatory role of lncRNA on CVB5 infection and replication,so as to provide clues for us to understand the molecular role of CVB5 infection-mediated diseases.