Profile Analysis Of Circular RNAs And Preliminary Study On Function Of Hsa＿circ＿0070421 In Influenza A Virus Infected A549 Cells

Circular RNAs(circRNAs)are a novel type of non-coding RNAs(nc RNAs).Few of them can code proteins.Unlike linear RNAs terminated at 5′ end caps or 3′ poly(A)tails,circRNAs are featured by covalently closed loop structures without open terminals,thus appearing to be highly stable nuclease resistance.Initially,circRNAs were misinterpreted as byproducts of aberrant RNA splicing.Accumulating evidence has proved that circRNAs play important roles in the regulation of various biological processes,including host-virus interaction.Influenza A viruses(IAVs)are negative strand RNA viruses that belong to the family of Orthomyxoviridae.As highly contagious zoonotic pathogens,IAVs constantly circulate in a wide variety of species,including avian and mammalian animal,which pose a significant threat to human,domestic animal and wildlife health.Though IAV has been studied intensively,our understanding of the regulatory function of circRNAs in IAV infection is still limited.The objective of the present study was to investigate the effect of IAV infection on circRNA expression profiles of A549 cells,and reveal the function roles of hsa＿circ＿0070421 in IAV infection.Firstly,we compared circRNA expression profiles in A549 infected with IAV(H1N1/PR8).Circ RNAs were considered as differentially expressed when differences in expression levels.Of these circRNA changes,207 were significantly upregulated and166 were significantly downregulated to the IAV/mock comparison.The potential target genes of top ten differentially expressed circRNAs were predicted.To dig out the potential biological events behind the data and provide a broad overview of circRNA function,we conducted Gene Ontology and KEGG pathway enrichment analysis.Our results also showed that multiple circRNAs were related to the pathway of signal transmission,cancer,chemokines,cell proliferation and apoptosis et al,and the biologic function of transcriptional regulation,activity of poised RNA polymerase II,metabolism,nervous system et al.In addition,to validate the results from microarray results,we randomly detected five circRNAs and quantified using q PCR.Secondly,to determine whether IAV infection is closely related to hsa＿circ＿0070421 expression,we did experiments as follows:(1)Identifying the circular structure of hsa＿circ＿0070421;(2)We demonstrated that the expression of hsa＿circ＿0070421 was significantly induced by IAV infection in a dose-and timedependent manner;(3)hsa＿circ＿0070421 was found in all examined human cell lines,and its expression was observably increased after IAV infection;(4)hsa＿circ＿0070421expression was strongly induced by poly(I:C),not by LPS stimulation,cisplatin treatment,or serum withdrawal.(5)JAK-STAT pathway inhibitor(Ruxolitinib)significantly inhibited IAV-induced transcription of hsa＿circ＿0070421.These data suggest that IAV infection induces upregulation of cellular hsa＿circ＿0070421 through JAK-STAT activation and increment of hsa＿circ＿0070421 level is associated with viral RNA accumulation during replication.Finally,we wonder whether hsa＿circ＿0070421 reciprocally affects IAV replication.After transient overexpression of exogenous hsa＿circ＿0070421 and efficient inhibition of endogenous hsa＿circ＿0070421 by si RNA respectively,IAV replication was examined on RNA,protein and progeny virion levels.The results showed that forced expression of hsa＿circ＿0070421 inhibited IAV replication.In contrast,knockdown of hsa＿circ＿0070421 facilitated viral replication.The innate immune response of virusinfected cells is primarily governed by rapid production of type I IFNs,which confers an antiviral state to cells.We next investigated the effect of hsa＿circ＿0070421expression on type I interferon antiviral innate immune response.Overexpression of hsa＿circ＿0070421 enhanced IAV-induced Ifnb1 m RNA levels and ISRE promoter activation.Complementarily,the presence of hsa＿circ＿0070421 si RNA significantly repressed Ifnb1 transcription and ISRE promoter activity.These results suggest that hsa＿circ＿0070421 suppresses IAV replication in A549 cells through promoting IAVtriggered type I IFN signaling.Taken together,we describe here in a large data set of changes in the circRNA expressions of A549 cells infected with IAV,which will be useful for better understanding of the intricate interaction between IAV and host.Moreover,we also demonstrate that hsa＿circ＿0070421,as a host antiviral factor,play a role in suppression of IAV replication through positively regulates type I IFN signaling.In the future,the role of lots of differential expression circRNAs in IAV infection should be further explored.