Background Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). Methods We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs. Results The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation. Conclusion Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.

Objective Our study aimed to elucidate the regulatory molecules related to the osteogenic differentiation of periodontal ligament cells (PDLCs). Background Periodontal ligament cells are a favorable source for cell‐based therapy in periodontal bone engineering and regeneration due to their potential multilineage differentiation ability. However, the molecular mechanism and signaling pathways related to the osteogenic differentiation of PDLCs are still unclear. Methods Osteoblast‐specific protein expression levels were examined by ELISA in osteogenic‐induced PDLCs (induced‐PDLC group). A microarray assay and a bioinformatics analysis were carried out to reveal significantly expressed genes and the related pathways in induced‐PDLCs, and these findings were then confirmed by qRT‐PCR and a luciferase reporter assay. Finally, overexpressing and silencing gene systems were established to identify the specific transcriptional relationship and function of the target genes on the osteogenic differentiation of PDLCs. Results Osteogenically differentiated PDLCs with high levels of osteoblast‐specific proteins were established. The upstream stimulatory factor 2 (USF2) and activating transcription factor 4 (ATF4) mRNA levels were upregulated the most through the MAPK signaling pathway in the induced‐PDLC group. USF2 could bind to the transcriptional initiation region of ATF4 and regulate its transcriptional activities. Additionally, the overexpression of USF2 promoted osteoblast‐specific gene expression and the Alizarin red staining of PDLCs, while simultaneously overexpressing USF2 and silencing ATF4 reversed the favorable osteogenic effect of the induced‐PDLCs by reducing osteoblast‐specific gene expression and the Alizarin red staining level. Conclusion Our study demonstrated that USF2 could enhance the osteogenic differentiation of PDLCs by regulating ATF4 transcriptional activities, which provides a new strategy to utilize USF2 and ATF4 as potential target molecules for periodontal bone regeneration.

Objective Regulated cell death is key in the pathogenesis of persistent apical periodontitis. Here, we investigated the mechanisms of regulated cell death in osteoblast‐like MG63 cells infected with Enterococcus faecalis OG1RF. Materials and methods MG63 cells were infected with live E. faecalis OG1RF at the indicated multiplicity of infection for the indicated infection time. We evaluated the cells by flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling assay and lactate dehydrogenase release analysis; measured the activity of caspase‐1/‐3/‐8/‐9 and the release of interleukin‐1β; and determined the expression of apoptosis‐associated proteins and gasdermin D by apoptosis antibody array and Western blotting. Results Enterococcus faecalis OG1RF reduced the mitochondrial membrane potential of the infected cells, increased the percentage of apoptotic and terminal deoxynucleotidyl transferase dUTP nick end labelling‐positive cells, and enhanced lactate dehydrogenase release. The expression of caspase‐3 and survivin and the activity of caspase‐3/‐8/‐9 were upregulated, while the expression of death receptor 6 was downregulated. The activity of caspase‐1/gasdermin D and the release of interleukin‐1β remained unaltered. Conclusion Enterococcus faecalis OG1RF induced both intrinsic and extrinsic MG63 cell apoptosis via caspase‐3/‐8/‐9 activation but did not activate the pyroptotic pathway regulated by caspase‐1/gasdermin D.