The Neuraminidase Fragment From Avian Influenza Virus H9N2 Regulates MiRNA And Actives The Immune Function Of Dendritic Cells

Avian influenza,a serious pathogen,causes acute respiratory disease infected by avian influenza virus(AIV).In recent years,frequent outbreaks of avian influenza have caused great economic losses to poultry industry in many countries.H9N2,a low pathogenic subtype AIV,is highly variable and can recombine with other subtypes of influenza viruses easily.Moreover,it often leads to increased virulence and causes outbreaks of influenza spread across species.The outbreak of H7N9 and H10N8 in China are derived from the recombination between H9N2 and other influenza virus.Therefore,it's urgent to search effective anti-influenza virus targets or drugs.The pathogenicity of avian influenza virus not only depends on the characteristics of the virus,but also rely on the host immune response.Dendritic cells(DCs),the most professional antigen presenting cells(APCs),play a core role in innate immunity and adaptive immunity,which also participates in the replication inhibiting of influenza virus.Nevertheless,non-coding RNA,regulatory RNA is involved in the regulation of development,maturation and antigen presentation functions of DCs.Particularly,recent studies have found that microRNA can influence the ability of DCs to respond to influenza viruses.Therefore,we are trying to explore the function of regulating dendritic cells at the level of non-coding RNAs,which provides an important theoretical basis for combating with AIV by miRNAs.In this study,three segments encoding the polymerase proteins NA,M and NS was focused,in which NA participated in cutting sialic connection and releasing progeny viruses.Firstly,we constructed the eukaryotic expression vectors of NA,M,NS and transfected the constructed vector into DCs.Results showed that the Mean fluorescence intensity(MFI)of CD80 and MHCII increased significantly when stimulated by NA,which indicated that NA can stimulate DC maturation and significantly affect the immune function of DCs.Secondly,the miRNA of three segments stimulated groups were extracted and then verified by qRT-PCR.Results displayed that the NA segment hugely up-regulated the expression of miR-155(fold change 4.67)and miR-674(fold change 2.12).Furthermorre,we continuted to study the interaction between miR-155 or 674 and DCs by overexpressing or interferencing the miRNA.Results demonstrated that overabundance of miR-155 and miR-674 increased the MFI of co-stimulatory CD40 and MHCII,while the increased expression of co-stimulatory molecules was totally blocked by inhibiting endogenous miR-155 and miR-674.Finally,to study the mechanism of action by miR-155 and miR-674,target genes of miR-155 and miR-674 were predicted and valided.Luciferase assays suggested that CAMK1D and MBNL3 groups significantly decreased the luciferase activity(P<0.05)in miR-674 treated group.The relative luciferase inhibition rate was 85.2%(CAMK1D)and 90.5%(MBNL3)respectively.Similarly results were observed in miR-155 and its target gene MYOID.Co-transfection of MYOID with miR-155 resulted in a huge decrease in luciferase activity.The relative luciferase inhibition rate was 58.2%.In conclusion,we constructed the direct gene interaction network of NA-miR155-MYOID,NA-miR674-CAMK1D NA-miR674-MBNL3.This network revealed potential important functions that miRNAs have in host and pathogen interactions.