MicroRNA miR-27a-5p reduces intestinal inflammation induced by Clostridioides difficile flagella by regulating the NF-κB signaling pathway

Background Clostridioides difficile is a major cause of nosocomial postantibiotic infections, often resulting in severe inflammation and watery diarrhea. Previous studies have highlighted the role of C. difficile flagellin FliC in activating Toll-like receptor 5 and triggering nuclear factor–κB (NF-κB) cell signaling, leading to the release of proinflammatory cytokines. However, the microRNA (miRNA)–mediated regulatory mechanisms underlying the FliC-induced inflammatory response remain unclear. Methods miRNA expression levels were analyzed in Caco-2 intestinal epithelial cells following FliC stimulation and infection with the epidemic C. difficile R20291 strain or its unflagellated mutant by reverse transcription–quantitative polymerase chain reaction. Chemical inhibitors were used to block NF-κB signaling, and their impact on miR-27a-5p expression was assessed. Knockdown and overexpression experiments with miRNA inhibitor and mimic respectively were conducted to elucidate the functional role of miR-27a-5p in FliC-induced inflammatory responses. Additionally, a mouse model of C. difficile infection was treated with miR-27a-5p to evaluate its therapeutic potential in vivo. Results miR-27a-5p showed significant FliC-dependent overexpression in Caco-2 cells. Inhibition of NF-κB signaling suppressed miR-27a-5p overexpression. Knockdown of miR-27a-5p increased NF-κB activation and cytokine production (tumor necrosis factor α and interleukin 8), while its overexpression had the opposite effect. Moreover, miR-27a-5p was overexpressed in the ceca of C. difficile–infected mice, correlating with intestinal interleukin 8 levels. Treatment of infected mice with the miR-27a-5p mimic reduced disease severity and intestinal inflammation. Conclusions miR-27a-5p plays a crucial role in regulating C. difficile–induced inflammation, suggesting its potential as a therapeutic target for controlling severe infection. These findings offer valuable insights into potential therapeutic strategies for managing C. difficile infection and associated inflammatory complications.