MiR-324-3p Down-regulates Coxsackievirus B3 Replication By Targeting Trim27 And Underling Mechanism

Background:Viral myocarditis is a myocardial disease caused by virus.It is more common in children.Most patients with this disease have a good prognosis,and a few can develop dilated cardiomyopathy,causing acute heart failure,cardiogenic shock and even sudden death.There are a variety of viruses that can cause viral myocarditis,of which Coxsackievirus B3(CVB3)is the virus more likely to cause viral myocarditis.CVB3 is a member of the Enterovirus genus in the Picornaviridae family.The genome of CVB3 is a positive,single-stranded RNA molecule encoding a single open reading frame(ORF)MicroRNA(miRNA)are non-coding small-molecule single-stranded RNAs of approximately 18-25 nucleic acids in length in eukaryotes,through the 3’-UTR specific sequence of the target gene.The form of complete or incomplete complementation allows the target gene mRNA to degrade or inhibit translation,and to reverse the role of the target gene,and the miRNA forms a complex post-transcriptional regulatory network to regulate a variety of cellular processesObjective:This study was to explore the role of miR-324-3p and their target genes in the regulation of CVB3 replication by exploring the differential expression of miR-324-3p during CVB3 infection and to understand its mechanism of action,and further clarify its regulatory mechanism for CVB3 replication.It can provide theoretical basis and molecular target for the diagnosis and treatment of CVB3 infectionMethods:1.The 3C was expressed by E.coli prokaryotic expression system and rabbit polyclonal antibody preparation was performed2.RT-qPCR to analysis the expression of miR-324-3p on mock,CVB3 infection HeLa cells after 5h and 8h3.CVB3 was infected after overexpression or silencing of miR-324-3p on HeLa cells The CVB3 genome replication was analyzed by RT-qPCR at 5 h and 8 h after infection,and the expression of viral protein was analyzed by Western blot.In addition,the plaque assay to analyze the mR-324-3p effect on the proliferation of CVB34.The miR-324-3p target gene was screened by miRTarBase and TargetScan databases and verified by luciferase reporter vector5.RT-qPCR and western blot to analysis trim27 expression on mock,and CVB3 infected HeLa at 3h,4h,5h,6h,8h.Overexpression or silence of trim27 in HeLa cells,collected protein after CVB3 infection for 5h and 8h,and mock was used as control.Western blot was used to detect the regulation of trim27 on CVB3 replication.At the same time,the virus plaque assay to analyze the regulation of trim27 on CVB3 proliferation6.Overexpression/silencing of miR-324-3p or trim27 in HeLa,collection mock,5h and 8h total protein,then use Western blot analysis of inflammatory-related signaling pathway cytokines NF-κB,p-NF-κB and IKKα/βResults:1.The 3C protein was successfully expressed in E.coli,and rabbit polyclonal antibody was preformed by immunizing New Zealand white rabbit2.After HeLa after CVB3 infection,Western blot can detect VP1 after 4 hours of infection,and 3C can be detected in 5 hours3.After overexpression of miR-324-3p,the expression of VP1 and 3C was decreased at 5h and 8h after infection with CVB3;when miR-324-3p was knocked down in cells,the expression of VP1 and 3C were increased at 5h and 8h after infection with CVB3.The results of virus plaque assay showed that miR-324-3p overexpression could reduce the production of viral plaques.On the contrary,after inhibiting miR-324-3p in cells,the number of viral plaques was more4.MiRNA target gene prediction databases found that miR-324-3p targets the 3’-UTR of trim27.The luciferase assay found that fluorescein expression was decreased compared with the target group,while the luciferin expression in the mutant group was observed There was no significant decrease compared with the control group,and the results indicated the targeted regulation of miR-324-3p on trim275.Infected CVB3 after overexpression of trim27 in HeLa cells,it was found that the expression of VP1 and 3C protein was increased at 5h and 8h after CVB3 infection.Virus plaque assay also found that overexpression of trim27 can increase the production of viral plaques;trim27 was knocked down in HeLa cells,and the expression of viral proteins VP1 and 3C was decreased at 5h and 8h after infection with CVB3.The number of viral plaques was also less than that of the control group6.Overexpression of miR-324-3p reduced the expression of IKKα/β and p-NF-κB.The expression of IKKα/β and p-NF-κB was enhanced when miR-324-3p was silenced in cells The expression of p-NF-κB,NF-κB and IKKα/β was lower than NC group when trim27 silenced.In addition,the infection was overexpressed with trim27.The expression of IKKα/β,NF-κB and p-NF-κB were higher than that of the controlConclusion:MiR-324-3p targets trim27 to negatively regulates CVB3 replication and proliferation.