Abstract
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-β 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.
Highlights
Odontogenic differentiation of human dental pulp stem cells is a key step of pulp regeneration
A variety of stem cells have been isolated from oral tissues, such as human dental pulp stem cells [5], stem cells from apical papilla (SCAPs) [6], periodontal ligament stem cells (PDLSCs) [7], dental follicle progenitor cells (DFPCs) [8], and stem/progenitor cells isolated from the human pulp of exfoliated deciduous teeth (SHED) [9]. hDPSCs are the most common dental-derived stem cells used in the study of odontogenic differentiation [10]: they originate
The culture media were renewed every 3 days. hDPSCs of passages 3–5 were used in follow-up experiments [29, 30], respectively, cultured in essential and odontogenic medium. hDPSCs in the noninduced were cultured in 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) with no supplements. hDPSCs in the induced group were cultured with an odontogenic differentiation medium containing 50 mg/ml ascorbic acid, 100 nmol/l dexamethasone, and 10 mmol/l β-glycerophosphate (Sigma, St Louis, Mo, USA) in DMEM for 14 days [31, 32]
Summary
Odontogenic differentiation of human dental pulp stem cells (hDPSCs) is a key step of pulp regeneration. 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. Bioinformatic analysis of the expression data suggested that circRNA-miRNAmRNA networks might act as a crucial mechanism for hDPSC odontogenic differentiation, providing a theoretical foundation for the study of pulp regeneration regulation by circRNAs. Pulpal and periapical diseases are the most common oral diseases: the traditional treatment of choice is root canal therapy. Many groups have been studying the odontogenic differentiation [13,14,15], the exact molecular mechanisms are still poorly understood, and the regeneration of dentin-pulp complex in the clinical context is still not implemented [16]. The search for new biomarkers linked to the molecular mechanisms of odontogenic differentiation is extremely important for studying pulp regeneration
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