MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts

Background: Although various microRNAs (miRNAs) regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated. In this study, we measured miRNA-146 (miRNA-146a and miRNA-146b-5p) expression and explored its regulatory function in the inflammatory response in human gingival fibroblasts (HGFs). Methods: miRNA-146a and miRNA-146b-5p expression was measured by performing real-time polymerase chain reaction in HGFs after Porphyromonas gingivalis (p.g) lipopolysaccharide (LPS) stimulation. After the HGFs were transfected with miRNA-146a and miRNA-146b-5p inhibitor, the expression levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by western blot and quantitative PCR. A luciferase assay was used to detect whether miRNA-146 could directly bind to the 3'-UTR of IRAK1. Results: The expression levels of miRNA-146a and miRNA-146b-5p significantly increased in the P.g LPS-stimulated HGFs compared to the non-stimulated HGFs. The inhibition of miRNA-146a and miRNA-146b-5p resulted in increased IL-1 beta, IL-6 and TNF-alpha secretion. The mRNA and protein levels of IRAK1, but not TRAF6, also increased. We further found that miRNA-146a and miRNA-146b-5p directly bound to the IRAK1 3'-UTR. Conclusion: Our data suggest that miRNA-146 inhibits pro-inflammatory cytokine secretion through IRAK1 in HGFs, which indicates that miRNA-146 functions as a negative regulator of periodontal inflammation.