miRNA-146 negatively regulates the production of pro-inflammatory cytokines via NF-κB signalling in human gingival fibroblasts

Objective: In human gingival fibroblasts (HGFs), TLR4 recognises Pathogen-associated molecular patterns (PAMPs), such as LPS, and subsequently activates downstream signals that lead to the production of pro-inflammatory cytokines. The aim of this study was to explore the mechanisms of LPS-induced miRNA-146 regulation of TLR4 signals in HGFs. Materials and methods: HGFs were treated with Porphyromonas gingivalis (P.g) LPS, the cells were harvested, and kinase phosphorylation levels were detected by western blot. Selective pharmacological inhibitors and agonists were used to block or activate the relevant kinases, miRNA-146a/b expression levels were detected by real-time PCR, and IL-1, IL-6, and TNF-alpha production were measured by enzyme-linked immunosorbent assays (ELISA). A luciferase reporter plasmid containing miRNA-146a/b promoter was tested in terms of p50/p65 regulation. Results: After P.g LPS treatment, NF-kappa B and Erk1/2 were strongly activated in HGFs. miRNA-146a/b, IL-1, IL-6 and TNF-alpha levels were down-regulated when NF-kappa B inhibitor was used. p50/p65 strongly activated miRNA-146a/b promoter as measured with the luciferase assay. Conclusion: In TLR4 signalling in HGFs, both miRNA-146a and miRNA-146b are downstream targets of NF-kappa B, but not of AP-1 signalling. miRNA-146a/b expression was specifically dependent on NF-kappa B but not Erk1/2 or JNK signalling.