The suppressive effects of miR-508-5p on the odontogenic differentiation of human dental pulp stem cells by targeting glycoprotein non-metastatic melanomal protein B

Abstract

BackgroundAlthough 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.MethodsIn 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.ResultsWe 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.ConclusionsOur 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.

Epigenetic Regulation of Dental Pulp Stem Cell Fate

“…In addition, during odontogenesis of DPSCs, miR-508-5p is gradually decreased, while glycoprotein nonmetastatic melanoma protein B (GPNMB), also called osteoactivin, is increased. Further research demonstrated that knockdown of miR-508-5p can promote odontogenesis in DPSCs via upregulation of GPNMB [ 159 ]. In conclusion, miRNAs affect the osteo/odontogenic differentiation of DPSCs by regulating various key molecules in the osteo/odontogenesis process.…”

Circular RNA SIPA1L1 regulates osteoblastic differentiation of stem cells from apical papilla via miR-204-5p/ALPL pathway

“…According to reports, Ingenuity® Pathway Analysis results showed that miR-204-5p can directly target ALPL [ 28 ]. Here, the bioinformatics analysis and luciferase reporter experiments further proved that miR-204-5p targets the 3′UTR of the ALPL gene, inhibiting translation of ALPL at the post-transcriptional level.…”

“…On the other hand, multiple indispensable genes related to osteoblasts differentiation and bone mineralization have been revealed. Among these genes, ALPL (alkaline phosphatase) which is recognized as an early expression gene associated with osteogenesis has attracted our attention [ 28 ].. The alkaline phosphatase (ALP) protein it encodes has been widely used as a diagnostic indicator to assess the grade osteoporosis [ 29 ].…”

Multidifferentiation potential of dental-derived stem cells

“…Additionally, during odontogenesis of hDPSCs, the expression of miR-508-5p decreases gradually, while significant inhibition of odontogenesis is observed after overexpression of miR-508-5p, which targets glycoprotein nonmetastatic melanomal protein B[ 120 ]. Xu et al[ 121 ] reported that during odontoblast differentiation of DPSCs, the expression of miR-21 can be regulated by treating with TNF-α, while downregulation of miR-21 causes a decrease in the expression of dentine matrix protein 1 and DSPP by interacting with STAT3[ 121 ].…”