Pre-treatment with AgomiR-93 inhibits pro-inflammatory gene expression in Pseudomonas aeruginosa-infected Cystic Fibrosis IB3 1 bronchial epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by mutations in the CFTR gene, persistent airway inflammation, and recurrent Pseudomonas aeruginosa (P. aeruginosa) infections. In CF, MicroRNAs (miRNAs) play a key regulatory role in both the hyperinflammatory state and the host response to bacterial pathogens. This study investigated whether pre-treatment of CF bronchial epithelial IB3-1 cells with AgomiR-93-5p modulates the inflammatory response induced by P. aeruginosa and explored potential underlying mechanisms. Methods: IB3-1 cells were treated with AgomiR-93-5p for 48 hours prior to infection with P. aeruginosa. Expression of IL-6, IL-8, G-CSF, and IL-1β mRNAs was quantified by RT-qPCR. Cytokine release was assessed by Bio-Plex multiplex analysis. Effects on the accumulation of mRNAs conding NF-κB p50 and p65 subunits, TLR4, IRAK4 were also evaluated. Pre-treatment with AgomiR-93-5p significantly reduced the P. aeruginosa-induced upregulation of IL-6, IL-8, G-CSF, and IL-1β at both mRNA and protein levels. A marked downregulation of the NF-κB p50 and IRAK4 transcripts was also observed. The inhibitory effect on IL-8 expression is consistent with a predicted direct interaction between mature miR-93-5p and the IL-8 3′UTR. Additional effects on IL-6, IL-1β, and G-CSF may be mediated by inhibition of the TLR4/MyD88/NF-κB signaling pathway, supported by predicted miR-93-5p binding sites within the 3′UTRs of TLR4 and IRAK4. AgomiR-93-5p effectively mitigates P. aeruginosa-driven inflammatory responses in CF bronchial epithelial cells, acting through both direct cytokine mRNA targeting and modulation of TLR4-dependent NF-κB signaling. miRNAs regulating this pathway represent promising therapeutic candidates for chronic inflammatory diseases such as cystic fibrosis.