bioRxiv2024
AimTo investigate the effect of microRNA-27a-5p (miR-27a-5p) on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). MethodologyExpression of miR-27a-5p was evaluated in LPS-stimulated hDPCs isolated from third molars of healthy patients by a TaqMan microRNA assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and a cytometric bead array were used to measure mRNA and protein expression levels, respectively, of proinflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein 1 (MCP1). Luciferase assays and western blotting were conducted to assess nuclear factor {kappa}B (NF-{kappa}B) activity. Gene/protein expression of NF-{kappa}B signaling activators, i.e., transforming growth factor beta-activated kinase 1 binding protein 1 (TAB1), IL-1 receptor-associated kinase 4 (IRAK4), NF-{kappa}B p65 (RELA), and follistatin-like 1 (FSTL1), was evaluated by RT-qPCR and western blotting. Transfection of an miR-27a-5p mimic was performed using Lipofectamine RNAiMax reagent. Wildtype and mutated 3{square}-untranslated region (UTR) of TAB1-contained luciferase reporter vectors were co-transfected with the miR-27a-5p mimic. Small interfering RNA against TAB1 (siTAB1) was designed to block its expression. One-way analysis of variance and Students t-test were used to determine statistical significance ( = .05). ResultsLPS-stimulated hDPCs showed concurrent increases in the expression of miR-27a-5p and proinflammatory cytokines (IL-6, IL-8, and MCP1), and the increased expression was suppressed by NF-{kappa}B inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated expression of proinflammatory cytokines, NF-{kappa}B activity, and expression of NF-{kappa}B signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3{square}-UTR of TAB1. siTAB1 downregulated NF-{kappa}B activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-{kappa}B activity, and NF-{kappa}B signaling activator expression (TAB1, IRAK4, and RELA) was also found in LPS-stimulated rat incisor pulp tissue explants following transfection with the miR-27a-5p mimic ex vivo. ConclusionsMiR-27a-5p, whose expression was induced by NF-{kappa}B signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-{kappa}B signaling. In particular, TAB1, a potent NF-{kappa}B activator, was targeted by miR-27a-5p. These results provide insights into the negative regulatory effects of miR-27a-5p, particularly those targeting the TAB1-NF-{kappa}B signaling pathway, on pulp inflammation.