| Influenza A virus(IAV)is the agent of influenza outbreaks,from the Spanish flu in 1918 to the swine flu in 2009,and to seasonal flu in 2018,tens of millions of people have died,countless domestic fowls have been killed from the flu outbreak.It has been a major threat to human health and breeding industry.With the rapid development of highthroughput sequencing techniques and biotechnology,the important biological functions of circRNA are gradually recognized by researchers.However,their role in the infection of IAV is largely unknown.1.Analysis of the expression profile of non-coding RNA in A549 cells infected with influenza virus H1N1The expression levels of circRNA,mRNA and miRNA in A549 cells infected with A/PuertoRico/8/1934H1N1(H1N1)were detected by high throughput sequencing techniques.The results showed that A total of 412 circRNAs,7451 mRNA transcripts,and 91 miRNAs were significantly dysregulated after H1N1 virus infection.As expected,the expression level of these selected circRNAs,mRNA and miRNA candidates was consistent with RNA-seq data.The combined analysis of circRNA,miRNA and mRNA showed that the differentially expressed circRNAs may be involved in the regulation of cellular biological processes through the circRNA-miRNA-mRNA regulatory axis.These genes regulated by circRNAs are significantly enriched in "apoptosis pathway","p53 signal transduction pathway","cell cycle pathway","RIG-Ⅰ-like receptor signal transduction pathway","NF-κB signal transduction pathway" and "Node-like receptor signal pathway"suggesting that circRNAs may be involved in regulating the pathogenic mechanism and cellular immune response of influenza virus.2.Circ-GATAD2A promotes H1N1 virus replication through inhibiting autophagyThe up-regulation of circ-GATAD2A in the process of H1N1 virus infection was selected for functional analysis.The results showed that knockdown of circ-GATAD2A in A549 cells enhanced autophagy and inhibited H1N1 replication.By contrast,overexpression of circ-GATAD2A impaired autophagy and promoted H1N1 virus replication.Similarly,knockout of vacuolar protein sorting 34(VPS34)blocked autophagy and increased H1N1 virus replication.However,the expression of circ-GATAD2A could not further enhance H1N1 virus replication in VPS34 knockout cells.Collectively,these data indicated that circ-GATAD2A promotes the replication of H1N1 virus by inhibiting autophagy.3.Circ-MDM2-1 promotes Influenza virus replication through regulating KPNB1A significantly upregulated circ-MDM2-1 after H1N1 virus infection was selected for analysis.The results showed overexpression of circ-MDM2-1 promoted the replication of H1N1,H3N2 and H9N2 virus in A549 cells.Knockout of circ-MDM2-1 inhibited replication of H1N1,H3N2 and H9N2 virus,indicating that circ-MDM2-1 promoted influenza virus replication.The results of nucleoplasmic separation and situ hybridization showed that circ-MDM2-1 was mainly located in the cytoplasm,indicating that circMDM2-1 played a role in the cytoplasm.Through the analysis of online doRiNA database,it was found that circ-MDM2-1 was bound to AGO2.Further experiments confirmed the interaction between circ-MDM2-1 and AGO2 protein,indicating that circ-MDM2-1 may function as miRNA sponge,and further confirmed the binding of circ-MDM2-1 to hsamiR-370-3p by software analysis and experimental verification.Overexpression of hsamiR-370-3p can inhibit the replication of H1N1,H3N2 and H9N2 virus,inhibition of hsamiR-370-3p promoted the replication of H1N1,H3N2 and H9N2 virus.It was further analyzed and verified that KPNB1 was the target gene of hsa-miR-370-3p,and RNA interference-mediated silencing KPNB1 inhibit H1N1,H3N2 and H9N2 virus replication.In conclusion,circ-MDM2-1 promoted KPNB1 expression and influenza virus replication by adsorbing hsa-miR-370-3p after influenza virus infection... |
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