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MiR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

ABSTRACT: Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation. Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A. Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs. Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

SUBMITTER: Wang N

PROVIDER: S-EPMC8591228 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Although the involvement of glycoprotein non-metastatic melanomal protein B (GPNMB) in regulating the odontogenic differentiation of human dental pulp stem cells (hDPCs) has been identified, the underlying mechanisms are largely unknown. The purpose of this study is to investigate the effects of miR-508-5p on the GPNMB expression and the odontogenic differentiation of hDPCs. METHODS:In this study, hDPCs were isolated and identified by flow cytometric analysis. Based on bioinformatics analysis, dual luciferase reporter assay was performed to verify GPNMB acting as a target of miR-508-5p. The regulatory roles of miR-508-5p in odontogenetic differentiation of hDPCs were investigated through its inhibition or overexpression (miRNA mimics and miRNA inhibitors). qRT-PCR and Western blot analysis were used to detect the expression of odontogenetic marker genes and proteins. The assays of alkaline phosphatase (ALP) activity and Alizarin Red S staining were performed to evaluate the odontogenetic phenotype. RESULTS:We first found that the levels of miR-508-5p expression decreased gradually during odontogenesis of hDPCs, while the expressions of GPNMB were upregulated obviously. The suppressive effects of miR-508-5p on GPNMB were determined by oligonucleotide transfection in hDPCs and dual luciferase reporter assay in 293T cells. Subsequently, the significant inhibition of hDPC odontogenesis after the overexpression of miR-508-5p was observed, which is consistent with the decreased expression levels of several odontoblast-specific genes, such as dentin matrix protein 1 (DMP-1), dentin sialophosphoprotein (DSPP), and osteocalcin (OCN), as well as the decreased activity of ALP and weakened Alizarin Red S staining. Furthermore, ectopic expression of GPNMB (lacking 3'-UTR) rescued the effects of miR-508-5p on odontogenic differentiation. CONCLUSIONS:Our study demonstrated that miR-508-5p regulated the osteogenesis of hDPCs by targeting GPNMB and provided novel insight into the critical roles of microRNAs in hDPC differentiation.

Project description:IntroductionOdontogenic 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.MethodsIsolated 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.ResultsWe 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.ConclusionThis 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.

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MiR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

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