MicroRNA-182 Inhibits HCMV Replication Through Activation Of Type Ⅰ IFN Response By Targeting FOXO3 In Neural Cells

BackgroundIn the herpes virus population,human cytomegalovirus(HCMV)is a major virus,which causes congenital developmental brain disorders and fatal brain damage in immunocompromised patients.Congenital HCMV infection may lead to a variety of neurological anomalies including mental retardation,visual defects and seizures.For immunosuppressed patients,HCMV infection can result in CMV related encephalitis/ encephalopathy,which may cause serious illness or even death.The brain is a major target in HCMV infection and HCMV preferentially infects neuronal cells,However,little is known about the mechanism of its replication in neuronal cells.Therefore,it is of great significance to study the replication mechanism of HCMV in nerve cells for understanding the neurological defects of nervous system caused by HCMV.MicroRNAs(miRNAs)are a class of small non-coding RNA molecules which is usually about 21–25 nucleotides in length.Previous studies have demonstrated that miRNAs are involved in many viral infections.For example,miR-146 was reported to suppress the interferon reduction via targeting TRAF6 to promote replication of dengue virus.Infection with influenza virus usually leads to modulation of microRNA profiles(e.g.miR-146 a and miR-155)and suppression of type I interferon(IFN)responses of host cell.It also has been found that miR-138 is involved in the regulation of HCMV replication in endothelial cells,while there are relatively few studies on the regulation of miRNAs on HCMV-infected nerve cells,which had aroused our great research interest.Immunomodulation plays a vital role in HCMV infected process.For people with normal immune function,the immune system can inhibit HCMV replication in host cells.However,in immunocompromised individuals,the immune system cannot suppress the infection process of HCMV.In addition,HCMV can also induce immune escape mechanism.HCMV can be hidden in the host for a long time.When the body immunity declines,the virus will replicate in large quantitiesi Aims1.To screen the microRNAs that was differently expressed before and after HCMV infection in neural cells;2.Explore the effects of miR-182 on HCMV replication in neural cells3.Identify the target gene of miR-182;4.Explore the molecular mechanism of miR-182 in regulating HCMV replication in neural cells.Content1.MicroRNA screening and miR-182 level detection;2.Regulation of miR-182 on interferon immune response and HCMV replication;3.Target gene prediction and identification of miR-182 and its intervention effect on miR-182 mediated interferon immune response and HCMV replication;4.Effects of miR-182 agomir on HCMV replication in animal models.Methods1.The data from accession: GSE33584 and GSE75305 were analyzed,and the most obvious 8 microRNAs(4 up-regulated and 4 downregulated miRNAs)were selected.2.The effects of different infection concentrations of HCMV on miR-182 level at different infection time in two nerve cells(U-251 MG and NPCs cell lines);3.After overexpression or interference with miR-182 in U-251 MG and NPCs nerve cells,the expression of HCMV copy number,virus titer and two key proteins of the virus(IE2 and pp65)were detected.4.After overexpression or interference with miR-182 in two nerve cells of U-251 MG and NPCs,the generation of endogenous IFN-α/β was detected.5 Transfected U-251 MG and NPCs cells with si-IFNAR1 and IFNAR2,and then detected the expression of interferon stimulating factor(ISGs),MxA and OAS1,and detected the copy number of HCMV and virus titer at the same time.6.Two prediction software(TargetScan and miRanda)were used to predict the target gene of miR-182,and the fork head box O3(FOXO3)was verified through the dual-luciferase reporter gene experiment.The protein expression changes of FOXO3 and its downstream gene IRF7 were detected in the miR-182 transfected into U-251 MG and NPCs before HCMV infection.7.Study on the function of target genes.FOXO3 overexpressed plasmid was constructed,and the plasmid was co-transfected with miR-182 mimics for overexpression,and the intervention effects of FOXO3 overexpression on miR-182 mediated interferon immune response and HCMV replication was detected.8.Agomir-182 was injected into mice through tail vein to overexpress it in vivo,and the effects of miR-182 overexpression on HCMV replication in mice was detected.Results1.HCMV infected cells lead to differences in the microRNA expression profile of host cells,among which miR-182 is one of the most significantly upregulated miRNAs,and this promoting effect was time and dose-dependent.2.Overexpressed miR-182 suppressed virus level,whereas miR-182 knockdown upregulated HCMV titer in U-251 MG and NPCs.3.Overexpression of miR-182 in U-251 MG and NPCs promoted type I interferon immune response,while silencing miR-182 inhibited type I interferon immune response.At the same time,we found that HCMV infection can induce up-regulation of ISGs,MxA and OAS1 expression,while interference with IFNAR1 and IFNAR2 can remove this promoting effects induced by HCMV,and more importantly,it can also remove the inhibition of miR-182 mimics on virus replication.4.We predicted that FOXO3 might be the target gene of miR-182.For this gene,we performed a dual-luciferase reporter assay and identified FOXO3 as a target of miR-182.On the other hand,we also found that miR-182 overexpression can inhibit FOXO3 expression and enhance the transcription activity of IRF7 in the case of virus infection.5.miR-182 overexpression promotes interferon immune response and inhibits HCMV replication.However,after simultaneous transfection of pcDNA-FOXO3,the antiviral function of miR-182 was inhibited.In contrast,the antiviral function of miR-182 was enhanced after si-IRF7 transfection.6.The overexpression of miR-182 in animal models also inhibited HCMV replication,and the level of FOXO3 and IRF7 was correspondly regulated.Conclusion1.HCMV infected neural cells significantly up-regulated the expression of miR-182.2.miR-182 overexpression can up-regulate the expression of IFN-α/β by modulating the expression of interferon receptors on the cell surface to activate the expression of ISGs,MxA and OAS1,thereby inhibiting HCMV replication in neural cells.3.miR-182 exerts antiviral function via activating the interferon immune response by targeting FOXO3 and up-regulating IRF7 in neural cells.4.Overexpression of miR-182 in animal models also inhibited HCMV replication.