Influenza virus induced novel miRNAs regulate the STAT pathway

MicroRNAs are essential regulators of gene expression in humans and can control pathogenesis and host-virus interactions. Notably, the role of specific miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza virus induced several novel miRNAs that were identified using next generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. Expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells (HBEpC) exposed to H1N1, H9N1(1P10) and H9N1 (1WF10) strains of influenza virus. A time course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10) infected cells, consistent with the NGS data. Additionally put-miR-34, and put-miR-35 showed a high fold enrichment in argonaute-immunoprecipitation compared to the controls, indicating their ability to form a complex with argonaute protein and RNA induced silencing complex (RISC), a typical mode of action found with miRNAs. Our earlier studies have shown that replication and survival of influenza virus is modulated by certain transcription factors, such as, NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed significant decrease in expression of Signal Transducers and Activators of Transcription 3 (STAT3), and the inhibitor of put-miR-34 showed significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region (UTR) of STAT3. NGS and PCR array data submitted to the Gene Ontology also predicted the role of transcription factors modulated by put-miR-34. Our data suggests that put-miR-34 could be a good target for the antiviral therapy as the hyperactivation or inactivation of STAT3 results in viral disease, as tightly regulated STAT3 function is central to health.