Project description:The roles of microRNAs (miRNAs) in odontogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unexplored. In this study, the underlying molecular mechanism of osteogenic differentiation in hDPSCs is investigated using miRNA profifiling. Overall design: Gene expression of hDPSCs at differentiated group were cultured with an odontogenic differentiation medium containing 50 mg/ml ascorbic acid, 100 nmol/L dexamethasone, and 10 mmol/L β-glycerolphosphate (Sigma, St Louis, MO, USA) for 14 days in 6-well plates. hDPSCs at undifferentiated group were cultured in 10 % FBS in DMEM with no supplements.
Project description:The roles of lncRNAs and mRNAs in odontogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unexplored. In this study, the underlying molecular mechanism of osteogenic differentiation in hDPSCs is investigated using miRNA profifiling. Overall design: Gene expression of hDPSCs at differentiated group were cultured with an odontogenic differentiation medium containing 50 mg/ml ascorbic acid, 100 nmol/L dexamethasone, and 10 mmol/L β-glycerolphosphate (Sigma, St Louis, MO, USA) for 14 days in 6-well plates. hDPSCs at undifferentiated group were cultured in 10 % FBS in DMEM with no supplements.
Project description:Introduction:Odontogenic differentiation of human dental pulp stem cells (hDPSCs) is a key step of pulp regeneration. Recent studies showed that circular RNAs (circRNAs) have many biological functions and that competing endogenous RNA (ceRNA) is their most common mechanism of action. However, the role of circRNAs in hDPSCs during odontogenesis is still unclear. Methods:Isolated hDPSCs were cultured in essential and odontogenic medium. Total RNA was extracted after 14 days of culture, and then, microarray analysis was performed to measure the differential expressions of circRNAs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was then performed to validate the microarray results. Based on microarray data from this study and available in the database, a ceRNA network was constructed to investigate the potential function of circRNAs during odontogenesis. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential correlation between signaling pathways and circRNAs. In addition, qRT-PCR and Western blot analysis were used to explore the function of hsa_circRNA_104101. Results:We found 43 upregulated circRNAs and 144 downregulated circRNAs during the odontogenic differentiation process (fold?change > 1.5 and <-1.5, respectively; P < 0.05). qRT-PCR results were in agreement with the microarray results. Bioinformatic analysis revealed that the Wnt signaling pathway and the TGF-? signaling pathway, as well as the other pathways associated with odontogenic differentiation, were correlated to the differentially expressed circRNAs. hsa_circRNA_104101 was proved to promote the odontogenic differentiation of hDPSCs. Conclusion:This study reported 187 circRNAs that were differentially expressed in hDPSCs during odontogenic differentiation. Bioinformatic analysis of the expression data suggested that circRNA-miRNA-mRNA networks might act as a crucial mechanism for hDPSC odontogenic differentiation, providing a theoretical foundation for the study of pulp regeneration regulation by circRNAs.
Project description:Osteo/odontogenic differentiation is a key process of human stem cells from apical papilla (SCAP) in tooth root development. Emerging evidence indicates microRNAs (miRNAs) play diverse roles in osteogenesis. However, their functions in osteo/odontogenic differentiation of SCAP require further elucidation. To investigate the role of miRNA in SCAP osteo/odontogenic differentiation and underlying mechanisms, miRNA microarray analysis was performed to screen differentially expressed miRNAs between control and osteo/odontogenic-induced group. Quantitative real-time PCR (qRT-PCR) and western blot were used to detected osteo/odontogenic differentiation-related markers and possible signaling pathway SCAP-associated genes. Alizarin Red Staining (ARS) were applied to evaluated osteogenic capacity. The results showed that miR-497-5p increased during SCAP osteo/odontogenic differentiation. Overexpression of miR-497-5p enhanced the osteo/odontogenic differentiation of SCAP, whereas downregulation of miR-497-5p elicited the opposite effect, thus suggesting that miR-497-5p is a positive regulator of the osteo/odontogenic differentiation of SCAP. Bioinformatic analysis and dual luciferase reporter assay identified that SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) is a direct target of miR-497-5p. Further study demonstrated that Smurf2 negatively regulates SCAP osteo/odontogenic differentiation, and silencing Smurf2 could block the inhibitory effect of the miR-497-5p inhibitor. Meanwhile, pathway detection manifested that miR-497-5p promotes osteo/odontogenic differentiation via Smad signaling pathway. Collectively, our findings demostrate that miR-497-5p promotes osteo/odontogenic differentiation of SCAP via Smad signaling pathway by targeting Smurf2.
Project description:Mesenchymal stem cells (MSCs) have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B (also known as JMJD3) was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate (ALP) activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 (osterix, OSX), as well as extracellular matrix genes BGLAP (osteoclacin, OCN) and SPP1 (osteopontin, OPN), was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 (BMP2) promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.
Project description:BACKGROUND:Bone marrow mesenchymal stem cells (BMMSCs) are suitable cell sources for dental pulp regeneration, but the mechanism of BMMSCs differentiation into odontogenic lineage remains unknown. The aim of the present study was to reveal the role of magnesium transporter protein 1 (MagT1) and MAPK pathways in the odontogenic differentiation of BMMSCs. METHODS:The RNA sequencing (RNA-seq) was performed to explore the altered transcriptome of BMMSCs undergoing odontogenic differentiation induced by tooth germ cell-condition medium (TGC-CM). Pathway analysis was conducted to explore enriched pathways of the differential expression signature. Automated western blot, real-time PCR, shRNA lentivirus, and flow cytometry were used to detect the function of MagTl and MAPK pathway in odontogenic differentiation of BMMSCs. RESULTS:RNA-seq identified 622 differentially expressed genes associated with odontogenic differentiation of BMMSCs induced by TGC-CM, some of which were responsible for MAPK pathway. Consistently, we verified that TGC-CM induced odontogenic differentiation of BMMSCs through activating ERK/MAPK pathway, and the inactivation of ERK/MAPK pathway inhibited the odontogenic differentiation induced by TGC-CM. We also found MagT1 protein was significantly increased during odontogenic differentiation of BMMSCs induced by TGC-CMM, in accordance, MagT1 knockdown significantly decreased the extent of mineralized nodules and the protein levels of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSP). Flow cytometry showed that intracellular Mg2+ was significantly reduced in MagT1-knockdown BMMSCs, indicating the suppression of MagT1 inhibited odontogenic differentiation of BMMSCs by decreasing intracellular Mg2+. Finally, we performed RNA-seq to explore the altered transcriptome of MagT1-knockdown BMMSCs undergoing odontogenic differentiation and identified 281 differentially expressed genes, some of which were involved in MAPK pathway. Consistently, automated western blot analysis found the ERK/MAPK pathway was inhibited in MagT1-knockdown BMMSCs during odontogenic differentiation, indicating that suppression of MagT1 inhibited odontogenic differentiation of BMMSCs via ERK/MAPK pathway. CONCLUSIONS:This study identified the significant alteration of transcriptome in BMMSCs undergoing odontogenic differentiation induced by TGC-CM. We clarified the pivotal role of MagT1 and ERK/MAPK pathway in odontogenic differentiation of BMMSCs, and suppression of MagT1 inhibited the odontogenic differentiation of BMMSCs by decreasing the intracellular Mg2+ and inactivating ERK/MAPK pathway.
Project description:BACKGROUND:Long noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS:hDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1). RESULTS:We identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1. CONCLUSION:This is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.
Project description:<h4>Background</h4>Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.<h4>Aims</h4>The aim of this study was to investigate nestin expression in various odontogenic tumours and evaluate its usefulness for histopathological diagnosis.<h4>Methods</h4>We studied formalin fixed, paraffin embedded specimens from 129 cases of odontogenic tumours and 9 of mandibular intraosseous myxoma. After characterisation of odontogenic ectomesenchymal tissues in these tumours using antibodies to vimentin, desmin, neurofilament, and glial fibrillary acidic protein, we immunohistochemically examined nestin expression.<h4>Results</h4>No differentiation towards muscle and nervous tissues was found in the odontogenic ectomesenchymal tissues. Although almost all the ameloblastomas and malignant ameloblastomas were negative for nestin, odontogenic ectomesenchyme in the odontogenic mixed tumours demonstrated nestin immunolocalisation, particularly in the region adjacent to the odontogenic epithelium. Odontoblasts and their processes, pulp cells near the positive odontoblasts, and flat cells adhering to the dentine showed immunoreaction with nestin in the odontomas and odontoma-like component in the ameloblastic fibro-odontomas. Neoplastic cells in almost half cases of jaw myxoma and one case of odontogenic fibroma expressed nestin.<h4>Conclusions</h4>The distribution of nestin in the odontogenic mixed tumours suggests that nestin expression in the odontogenic ectomesenchyme is upregulated by stimulation from odontogenic epithelium. In addition, nestin may also be involved in the differentiation from pulp cells to odontoblasts in odontogenic tumours. Therefore, nestin is a useful marker for the odontogenic ectomesenchyme and odontoblasts in odontogenic tumours. Nestin, one of the intermediate filaments constituting the cytoskeleton, is a marker of neural stem cells or progenitor cells. Its expression is also related to tooth development and repair of dentine.