Screening And Functional Study Of MiRNAs Involved In A549 Cells Infected With Influenza A Virus

Influenza virus is negative singale-strand RNA virus belonging to the Orthomyxoviridae family.Based on the different antigenicties of nucleice proteion and matrix protein,the influenza virus is mainly grouped into four classes(A,B,C,D).Influenza A virus(IAV)is the domainant pathogen of influenza,which is responsible for severe morbidity and mortality in animals and human worldwide.The antigenic drift and antigenic shift of influenza A virus make them the possibility of reassortment and escape to current various treatments,which is still a threat to human health.Thus,to prevent IAV infection,the development of new effective prophylactic or therapeutic drugs become urgently important.The virus-host interaction is very complex during influenza A virus infection.On the one hand,cellular factors may be used by IAVs for their own advantage.On the other hand,host factors can be involved in regulating innate and adaptive immune responds,acting as an antiviral defense mechanism.Micro RNAs(miRNAs)are a class of highly conserved,19-26 nt noncoding single-stranded RNA molecules that negative regulate gene expression by inducing mRNA degradation or inhibiting mRNA translation through binding to the 3' untranslated regions(UTRs)of their target genes.mi RNAs play important roles in a wide range of physiological and pathological processes,such as development,proliferation,differentiation,apoptosis,cancer,and viral infections.Recently,several studies indicated that miRNAs were involved in influenza A virus infection.However,the mechanism of these miRNAs is still not fully cleared.Therefore,we used two subtypes of influenza A viruses,A/Beijing/501/2009(H1N1/BJ501)and A/goose/jilin/hb/2003(H5N1/JH)to investigate the role of miRNAs during influenza A virus infection.This investigation may help us find a new basis for preventing and controlling the spread of flu.Firstly,A549 cells were mock infected or infected with A/Beijing/501/2009(H1N1)and A/goose/Jilin/hb/2003(H5N1)at a MOI of 5 for the indicated times.We observed the cytopathic effect(CPE)of infected cells to ensure efficient infection,and then dectected the level of antiviral mRNAs and the transcript of the influenza A virus polymerase subunit NP.We found the expression of all tested genes were significantly induced by IAV infection.Together,these results indicated permissive and productive infection of A549 by IAV.Subsequently,Microarray data and real-time PCR indicated that miR-141 and miR-200 c were up-regulated,while miR-21-3p and miR-29b-1-5p were downregulated during influenza A virus infection of human cells.However,the magnitude of fold-change occurring in H5N1 infection was stronger than that in H1N1 infection.Thirdly,we used the UV-treated IAV and a synthetic mimetic of viral doublestranded RNA,poly(I:C)to stimulate cells and found miR-141,miR-200 c,miR-21-3p and miR-29b-1-5p could not be regulated.These findings suggested that viral replication was essential for the regulation of four miRNAs expression.To identify target genes of these differentially expressed miRNAs during IVA infection,two computational algorithm programs,TargetScan and MicroCosm,were used to predict the target genes.Subsequently,GO and pathway analysis revealed that these targets were involved in fundamental cellular pathways and pathways associated with influenza A virus infection,such as Wnt signaling pathway and MAPK signaling pathway,and palyed an important role in IAV infection.Since influenza A virus could regulate the expression of miR-141,miR-200 c,miR-21-3p and miR-29b-1-5p,we next investigate whether these miRNAs affect IAV replication in turn.We first focused on miR-21-3p.Firstly,through dual luciferase reporter gene system,we found miR-21-3p could target eight potential genes and had the strongest inhibitory effect on the luciferase activity of pMIR-report vector linked to HDAC8 3'UTR.Furthermore,we found miR-21-3p regulated the expression of HDAC8 at both mRNA and protein levels.These results indicated that HDAC8 was a direct target of miR-21-3p.Subsequently,we used miR-21-3p mimic and inhibitor to investigate the effect of miR-21-3p on IAV replication.The results indicated that viral RNA of NP and viral titer were increased in infected cell at the presence of mi R-21-3p mimic,whereas miR-21-3p inhibitor significantly decreased IAV replication.At last,the roles of HDAC8 in IAV replication were validated by an RNAi strategy.We found the amount of NP and viral titer was increased in infected cells transfected with siHDAC8-1,which was consistent with the effect of miR-21-3p mimic.Together,these results demonstrated that miR-21-3p regulated the IAV replication,at least in part,via regulation of HDAC8 and host may resist influenza A virus infection through downregulation of miR-21-3p.Next,we turned the goal to miR-141 and miR-200 c.Firstly,we cloned the promoter of miR-141 and miR-200 c into pGL3 vector and found H1N1 and H5N1 could activate the promoter of miR-141~200c through dual luciferase reporter gene system.The results showed that influenza A virus induced the expression of miR-141 and miR-200 c at the transcriptional level.Secondly,through dual luciferase reporter gene system,we found miR-141 targeted three potential genes and miR-200 c targeted seven potential genes.Among these potential targets,the luciferase activity of pMIR-report vector linked to HDAC4 3'UTR was both repressed by miR-141 and miR-200 c,and the inhibitory effect on the luciferase activity of pMIR-report vector linked to XIAP 3'UTR by miR-200 c was most significant.Furthermore,we found miR-200 c negatively regulated the expression of XIAP at both mRNA and protein levels and miR-141 and miR-200 c downregualted the expression of HDAC4 at the protein levels.These results indicated that XIAP was a direct target of miR-200 c and HDAC4 was a common target of miR-141 and miR-200 c.Subsequently,we used miRNA mimic and inhibitor to investigate the effect of miR-141 or miR-200 c on IAV replication.The results indicated that viral RNA of NP and viral titer were decreased in infected cell at the presence of miR-200 c mimic,whereas miR-200 c inhibitor significantly increased IAV replication.Unfortunately,miR-141 had no significant effect on IAV replication.At last,the roles of XIAP in IAV replication were validated by an RNAi strategy.We only found the expression of H5N1 NP and viral titer were decreased in infected cells transfected with si XIAP-2,while XIAP siRNA had no significant effect on H1N1 replication.These results seemed to be inconsistent with the effect of miR-200 c mimic.Together,these results demonstrated that miR-200 c inhibited the IAV replication,incompletely,via regulation of XIAP and revealed the host may resist influenza A virus through upregulation of miR-200 c.In summary,our study reveals the host may resist influenza A virus H1N1 and H5N1 infection through upregulation of miR-200 c and downregulation of mi R-21-3p,which can provide a new evidence for the prevention and control the spread of flu.