Identification Of IRF5 As A Novel Target Of MiR-302a Involved In Influenza A Virus Infection

Influenza A virus H3N2 is a subtype of Influenza viruses,which belong to the Orthomyxoviridae family and classified according to the transmembrane proteins on the surface of virus coat:hemagglutinin(H)and neuraminidase(N).IAV H3N2,which has the most morbidity and mortality circulating in the human population,can also infect birds and pigs.By recombination of gene segments between different IAV,IAV H3N2 may strength its infectivity and cause an outbreak of local area or worldwide pandemic.The Hong Kong Flu was reassorted from IAV H3N2 and H2N2(Asian flu 1957)to form a new virus subtype by antigenic shift.The overwhelming pandemic of Hong Kong Flu in 1968 and 1969 caused one million death all over the world.Another novel influenza virus(2009 pdmH1N1)was reassorted from H3N2 and H1N1 virus and progressed to a pandemic in mid-2009.Therefore,the research of the characteristics and pathematology is very important for public health.The mainly targets of influenza viruses are the epithelial cells of the upper respiratory tract,and they can also infect macrophages and dendritic cells.The immune response of IAV infection requires both innate and adaptive immune systems.Through the reorganization of the Toll-like receptors(TLRs)and the retinoic acid-inducible gene 1(RIG-I),the IAV infected-cells are able to mediate induction and secretion both of the IFNs and pro-inflammatory factors as IL-6,IL-8 and TNF-? to resistant the virus replication.Meanwhile,the influenza virus have developed a novel mechanism to escape from the immunity system.For example,the NS1 protein encoded by influenza viruses is known to inhibit the activation of NF-?B and induction of interferon a and p.The main methods for this research are including to use the biological information technology to identify the target gene of miRNA according to their seed sequences.We constructed dual luciferase reporter plasmids containing the 3'UTR sequences of the target gene.And we co-transfect the plasmids with miRNA mimics or inhibitors.By Measuring and calculating the relative luciferase activity,we verify the post-transcription regulation of the gene by miRNA.There will be more experiments as fluorescence quantitative PCR and Western Blotting to further prove the results.With stimulation of IAV H3N2 or TLR agonist,we analyze the relative expression level of viral RNA,type I interference and inflammatory factors.In this assay,miR-302a was selected by the microarray analysis of microRNA expression in A549 cells after 4 hours infection with H3N2.Studies indicate that miR-302a up-regulating the transcription and translation of IAV H3N2 virus,and vice versa,down-regulating by miR-302a inhibitors.MiR-302a has been predicted to target IRF5,and it was determined by three methods described above.Previous reports indicate the role of IRF5 as an important regulation factor in virus suppression and type I interferences and inflammatory factors induction.MiR-302a mimics can promote the transcription and translation activities of IAV H3N2 by inhibiting the expression of IRF5.IRF5 is also a central mediator of toll-like receptor 7 signaling.Our results exhibited the miR-302a/IRF5 feedback regulation plays a crucial part of TLR7 singling pathway in A549 and THP-1 cell line.Briefly,this study was aimed at determining the molecular role and regulation mechanism of IRF5 in IAV H3N2 infected cells.And we investigated the function roles of miR-302 cluster in immune and inflammatory response by targeting some virus-induced pathway.