The Study On The Effect And Molecular Mechanism Of MicroRNA-203 In Influenza A Virus Infection

Influenza A virus(IAV),a negative single-stranded RNA virus belonging to the family Orthomyxoviridae,has a genome composed of eight segments.It can cause contagious respiratory infection with symptoms such as headache,fever and generalized muscular pain;in some severe cases,infection can prove fatal.Due to the highly variable antigenicity,wide host range,and high infectivity,the IAV easily leads to localized epidemic outbreak or even the global flu pandemic.Micro RNAs are a class of non-coding RNA molecules that are widely involved in various biological processes.There are complex interactions between host cellular miRNAs and viruses during viral infections.The expression of host cellular miRNAs is significantly altered in the influenza virus infection.The virus can utilize some miRNAs to achieve immune evasion,while the host can also trigger a series of anti-influenza virus responses through changes in miRNAs expression.Studying on the role of miRNAs in the influenza virus infection will further clarify the interaction between virus and host at the miRNA level.It can also help to understand the pathogenic mechanism of the virus and the host's anti-viral immune response better,providing new targets and strategies for development of novel anti influenza virus drugs.In this study,we aimed to identify candidate miRNAs that participate in host immune responses to IAV infection through microarray analysis of miRNAs and screened out miR-203 for further study.We preliminarily elucidated the molecular mechanism of IAV infection inducing miR-203 expression and the effects of miR-203 on IAV replication.A novel target gene of miR-203 called DR1 was also identified,which was involved in regulating the replication of IAV.The main contents of this study are as follows: 1.Screening and verification of miRNAs involved in IAV infectionTo identify candidate miRNAs that participate in IAV infection,we examined changes in the abundance of miRNAs in A549 cells after they were infected with A/Vietnam/1194/2004(H5N1)or A/Beijing/501/2009(H1N1,501)for 24 and 48 h through microarray analysis of miRNAs.After comparing of all these differentially expressed miRNAs in both virus infections at 24 and 48 h post-infection,eight common miRNAs were identified;of these,only miR-203 was up-regulated.To further verify the induction of miR-203 during IAV infection,A549 cells were infected with different subtypes of IAV(A/PR/8/34,A/Beijing/501/2009,A/Wisconsin/67/2005,A/Vietnam/1194/2004,and A/Anhui/01/2013)for 24 or 48 h,and alterations in expression of miR-203 were measured by quantitative real-time PCR.Meanwhile,dynamic changes in expression of miR-203 in A549 cells infected with H1N1(501)and H5N1 within 48 h were further analyzed.All of the results showed that miR-203 expression was significantly increased.So we chose miR-203 as candidate host cellular miRNA involved in IAV infection for further study.2.Molecular mechanism of miR-203 expression induced by IAV infection(1)The role of type?IFN in the induction of miR-203 during IAV infection.To investigate whether the up-regulation of miR-203 is related to IFN signaling during IAV infection,A549 cells were treated with IFN-? alone for 12 h and up-regulated miR-203 was detected.Then the promoter region of miR-203(2500bp)was amplified to construct p GL3-promoter vectors.A dual-luciferase assay was conducted revealing that IFN-? stimulated miR-203 promoter region activity directly.The analysis of sequence of the miR-203 promoter region showed a large number of transcription factor bingding sites associated with IFN signaling pathways.These indicated that IFN plays an important role in induction of miR-203 expression during IAV infection.(2)The effect of DNA demethylation in miR-203 promoter region in the induction of miR-203 during IAV infection.Expression of miR-203 was also up-regulated in H5N1 infected Vero cells,a cell line defective in IFNs production,indicating that factors other than type?IFN were also involved in induction of miR-203.Then we performed bisulfite sequencing PCR to examine DNA methylation modifications induced by IAV infection in miR-203 promoter region.The results showed that the CpG island(600bp)in the miR-203 promoter region in A549 cells carried fewer methylations when infected with H5N1 for 48 h.We further treated A549 cells and Vero cells with 5-aza-2'-deoxycytidine,a methylation inhibitor,and found increased expression of miR-203.The data showed that DNA demethylation was another mechanism in induction of miR-203 during IAV infection.DNA methylation is mainly mediated with three DNMTs: DNMT1,DNMT3 a,and DNMT3 b,of which expression of DNMT1 was down-regulated in H5N1 infected A549 cells.Furthermore,overexpression of DNMT1 inhibited induction of miR-203 during IAV infection while knockdown of DNMT1 could up-regulate miR-203 expression,demonstrating that DNA demethylation was caused by down-regulated DNMT1,which can further induce miR-203 expression in IAV infection.3.Effect of miR-203 on replication of IAVTo study the effect of miR-203 on IAV replication in depth,a miR-203 knockout(miR-203-KO)A549 cell line was constructed using the CRISPR/Cas9 system.Wild-type A549 cells and miR-203-KO cells were transfected with a miR-203 mimic,followed by infection with H5N1 virus.Virus growth curves were then generated and examined in a plaque assay.The results showed that miR-203 inhibited replication of IAV.Then we compared the abundance of viral nucleic acids in wild-type and miR-203-KO A549 cells at the early stage of virus infection,and found that the copy numbers of viral nucleic acid were higher in miR-203-KO cells than in wild-type cells,which further confirmed the inhibition of miR-203 on IAV replication.4.Molecular mechanism of miR-203 inhibiting IAV replication(1)Identification of miR-203 target genes involved in IAV replication.We treated wild-type A549 cells and miR-203-KO A549 cells with H5N1 for 48 h to measure the differentially expressed genes through mRNA expression profiling analysis.Meanwhile,the online tool TargetScan was used to predict biological targets of miR-203.By combining these two results,we identified 44 candidate target genes.To further identify one or more novel target genes,we transfected A549 cells for 48 h with the miR-203 mimic and measured the abundance of ten mRNAs(top ten differentially expressed genes).The results revealed a marked down-regulation in DR1(down-regulator of transcription 1)mRNA.Then,two sites matching the seed region of miR-203 in DR1 3'UTR region were amplified to construct pmirGLO vectors,and two mutant vectors were also constructed.The dual-luciferase assay data showed that miR-203 significantly reduced the luciferase activity of vectors containing 3'UTR region,however,the reduction was completely rescued by mutation of the seed match sequence.These results indicated that DR1 is one target gene of miR-203.(2)Effect of miR-203 on replication of IAV.A549 cells were transfected with si-DR1 to down-regulate the expression of DR1,followed by infection with H5N1 virus.Virus growth curves were then generated and examined in a plaque assay.The results showed that virus yields from cells transfected with si-DR1 were lower than in control cells.It demonstrates that miR-203 inhibits IAV replication by targeting DR1.In summary,this study demonstrates that IAV infection up-regulates host cellular miR-203 expression through two different mechanisms.First,type?IFN induced by IAV infection can directly stimulate promoter region of miR-203.Second,IAV infection leads to DNA demethylation of CpG islands in the miR-203 promoter region.Both of these regulations at the transcriptional level result in up-regulation of miR-203.We also identified a novel target gene of miR-203,DR1.MiR-203 inhibits IAV replication by targeting DR1.This study revealed the molecular mechanisms of interaction between miR-203 and IAV infection,and identified DR1 as a novel target gene of miR-203.It firstly elucidates the relationship between IAV and host cellular miR-203,and explains the mechanism that IAV infection induces the expression of miR-203 and the negative feedback regulation of miR-203 on viral replication.It also enriches the interaction between influenza virus and host at the miRNA level.Therefore,we expect that this study will provide new theoretical supports for the research on the pathogenesis of influenza virus and host anti-viral responses.