MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

MicroRNAs (miRNAs) play a crucial role in cell differentiation through epigenetic regulation of gene expression. In human dental pulp cells, we have identified miRNA-27a being upregulated under inflammatory conditions. Here, we aimed to examine whether (i) overexpression of miRNA-27a in human dental pulp stem cells (hDPSCs) enhances their odonto/osteoblastic differentiation via Wnt and bone morphogenetic protein signaling; and (ii) hDPSCs overexpressing miRNA-27a promote new bone formation in vivo. hDPSCs were cultured in osteogenic medium to promote differentiation. To examine the role of miRNA-27a, hDPSCs were transfected with either a miRNA-27a mimic to enhance or an inhibitor to suppress miRNA-27a expression. Odonto/osteoblastic differentiation was assessed by evaluating the expression of specific markers, Wnt and bone morphogenetic protein (BMP) signaling molecules, and mineralization capacity using RT-qPCR, western blotting, Alizarin Red S (ARS) staining, and alkaline phosphatase (ALP) activity. Potential miRNA-27a binding sites in the 3'UTRs of DKK3 and SOSTDC1 were identified via bioinformatics analysis and validated through the luciferase reporter assay. In vivo, miRNA-27a-overexpressing hDPSCs were seeded into collagen honeycomb scaffolds and implanted into mouse calvarial bone cavities to assess new bone formation. MiRNA-27a was highly upregulated in hDPSCs committed to odonto/osteoblastic differentiation. Overexpression of miRNA-27a led to increased expression of odonto/osteoblastic markers and enhanced mineralization capacity, while inhibition of miRNA-27a had the opposite effect. MiRNA-27a targeted DKK3, promoting β-catenin nuclear translocation and inhibiting SOSTDC1, which enhanced SMAD1/5 phosphorylation. Binding sites for miRNA-27a were identified in the 3'UTRs of DKK3 and SOSTDC1. In vivo, miRNA-27a-overexpressing hDPSCs promoted new bone formation in mouse calvaria bone cavities. Transfection of miRNA-27a in hDPSCs enhanced their odonto/osteoblastic differentiation by targeting DKK3 and SOSTDC1, thereby promoting the Wnt and BMP signaling. Transplantation of miRNA-27a-overexpressing hDPSCs promoted new bone formation in vivo. These findings deepen our understanding of the effects of miRNA on Wnt and BMP pathways and suggest a potential clinical application for miRNA-27a in promoting hard tissue regeneration, offering a promising therapeutic target for dental and craniofacial tissue reconstruction.

MicroRNA-27a-5p (miR-27a-5p) was significantly upregulated in dental pulp inflammation, yet its underlying mechanisms remain unclear. This study investigated the effect of miR-27a-5p on the expression of proinflammatory cytokines in human dental pulp cells (hDPCs) stimulated by lipopolysaccharide (LPS). 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 the expression of proinflammatory cytokines, NF-κB activity, and the 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. 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.

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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.

Dental pulp tissue, enclosed within rigid dentin, is susceptible to bacterial invasion via dentinal tubules, often leading to severe pulpal inflammation. This condition is typically associated with a hypoxic microenvironment, yet the mechanistic link between hypoxia and inflammation remains unclear. We identified a marked upregulation of microRNA-210 (miR-210) in human dental pulp cells (hDPCs) cultured under hypoxic conditions. This study investigated the role of miR-210 in modulating inflammation in lipopolysaccharide (LPS)-stimulated hDPCs. Hypoxic conditions and enforced expression of hypoxia-inducible factor 1α (HIF1α) significantly increased miR-210 levels. While LPS stimulation elevated proinflammatory cytokines (Interleukin-6, Monocyte Chemoattractant Protein-1, and Tumor Necrosis Factor Alpha) and activated nuclear factor-kappa B (NF-κB) signaling, miR-210 overexpression suppressed LPS-mediated cytokine production and NF-κB activity. Luciferase assays revealed that miR-210 targets and negatively regulates TGF-beta activated kinase 1 binding protein 1 (TAB1), a key upstream regulator of NF-κB. Transfection with an miR-210 mimic reduced TAB1 expression, NF-κB activation, and cytokine output in both LPS-stimulated hDPCs and rat pulp tissue ex vivo. Conversely, miR-210 inhibition enhanced TAB1 levels and inflammatory cytokine expression under hypoxic conditions. These findings suggest that miR-210 mitigates inflammation via the TAB1–NF-κB pathway, functioning as a negative feedback regulator. miR-210 may represent a promising therapeutic target for pulpal inflammation.