CircRNA expression profiling in H1N1-infected primary human tracheobronchial epithelial cells identifies candidate immune-related circRNAs validated in A549 cells

Influenza A virus (IAV) remains a persistent threat to global pandemics. Although previous studies have profiled circular RNA (circRNA) expressions following IAV infection, none have been conducted in human primary cells. Here, we used CIRIquant to analyse circRNA expression in RNA-seq datasets from H1N1 IAV-infected human tracheobronchial epithelial (HTBE) cells and validated selected circRNAs in A549-PB1 cells. We identified 10, 6, 73, 20, and 41 differentially expressed (DE) circRNAs at 3, 6, 12, 18, and 24 hours post infection (hpi), respectively. Notably, nine circRNAs were commonly upregulated at 12, 18 and 24 hpi timepoints. Functional enrichment analysis revealed that the parental genes of DE-circRNAs and the circRNA-targeted genes were associated with antiviral immune response and viral infection. In A549-PB1 cells infected with H1N1, we validated the circular nature and full-length of two circRNAs from DDX58 (circ DDX58/9 and /11) and one from PML (circ PML ) using RNase R digestion, and circRNA-rolling circle amplification. RT-qPCR confirm their upregulation upon H1N1 infection, corroborating our bioinformatics results in HTBE cells. Moreover, these circRNAs were also induced by Vesicular Stomatitis Virus and Sendai Virus, suggesting a potential common molecular response mechanism for virus infections. circ PML and circ DDX58/9 were predicted to regulate mRNA targets in a circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network. Overall, this study is the first to report circRNA expression profiles in H1N1-infected HTBE cells, with selected circRNAs validated in A549 cells, highlighting DE circRNAs potentially involved in host responses to IAV and warrant further functional investigation.