MicroRNA-27a-5p downregulates the expression of proinflammatory cytokines in lipopolysaccharide-stimulated human dental pulp cells via NF-κB signaling pathway
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Aim
To 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).
Methodology
Expression 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 κB (NF-κB) activity. Gene/protein expression of NF-κB signaling activators, i.e., transforming growth factor beta-activated kinase 1 binding protein 1 (TAB1), IL-1 receptor-associated kinase 4 (IRAK4), NF-κB 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□-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 Student’s t -test were used to determine statistical significance (α = .05).
Results
LPS-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-κB inhibitor BAY 11-0785. Transfection of the miR-27a-5p mimic downregulated expression of proinflammatory cytokines, NF-κB activity, and expression of NF-κB signaling activators (TAB1, IRAK4, RELA, and FSTL1) in LPS-stimulated hDPCs. Luciferase reporter assays revealed that miR-27a-5p bound directly to the 3□-UTR of TAB1. siTAB1 downregulated NF-κB activity and proinflammatory cytokine expression. Downregulation of proinflammatory cytokine expression, NF-κB activity, and NF-κB 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 .
Conclusions
MiR-27a-5p, whose expression was induced by NF-κB signaling, negatively regulated the synthesis of proinflammatory cytokines via targeting NF-κB signaling. In particular, TAB1, a potent NF-κB 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-κB signaling pathway, on pulp inflammation.
