Peifeng Han

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.

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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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.