Hsa-miRNA-143-3p regulates the odontogenic differentiation of human stem cells from the apical papilla by targeting NFIC.

AIM: To evaluate the effects of hsa-miRNA-143-3p on the cytodifferentiation of human stem cells from the apical papilla (hSCAPs) and the post-transcriptional regulation of Nuclear factor I-C (NFIC). METHODOLOGY: miRNA expression profiles in human immature permanent teeth and during hSCAP differentiation were examined. hSCAPs were treated with miR-143-3p overexpression or silencing viruses, and the proliferation and odontogenic and osteogenic differentiation of these stem cells, and the involvement of the NFIC pathway, were investigated. Luciferase reporter and NFIC mutant plasmids were used to confirm NFIC mRNA as a direct target of miR-143-3p. NFIC expression analysis in the miR-143-3p overexpressing hSCAPs was used to investigate whether miR-143-3p functioned by targeting NFIC. Student's t-test and chi-square tests were used for statistical analysis. RESULTS: miR-143-3p expression was screened by microarray profiling and was found to be significantly reduced during hSCAP differentiation (p < .05). Overexpression of miR-143-3p inhibited the mineralization of hSCAPs significantly (p < .05) and downregulated the levels of odontogenic differentiation markers (NFIC [p < .05], DSP [p < .01] and KLF4 [p < .01]), whereas silencing of miR-143-3p had the opposite effect. The luciferase reporter gene detection and bioinformatic approaches identified NFIC mRNA as a potential target of miR-143-3p. NFIC overexpression reversed the inhibitory effect of miR-143-3p on the odontogenic differentiation of hSCAPs. CONCLUSIONS: miR-143-3p maintained the stemness of hSCAPs and modulated their differentiation negatively by directly targeting NFIC. Thus, inhibition of this miRNA represents a potential strategy to promote the regeneration of damaged tooth roots.

MagT1 regulated the odontogenic differentiation of BMMSCs induced byTGC-CM via ERK signaling pathway.

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.