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 genecentered 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. (Continued on next page)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.

No abstract

The present study aimed to investigate the effects of vascular endothelial growth factor (VeGF) and insulin-like growth factor-1 (iGF-1) on the proliferation, migration and differentiation of human carious dental pulp stem cells (hcdPScs), and to elucidate the underlying mechanism(s). cell counting kit-8 assay was used to detect the effect of different concentrations of iGF-1 and VeGF on the proliferation of hcdPScs. Transwell assay was used to detect the migratory ability of the hcdPScs. alizarin red and alkaline phosphatase (alP) staining were used to detect the osteogenic ability of hcdPScs, whereas the angiogenic ability of the hcdPScs was tested by tube formation assay. reverse transcription-quantitative polymerase chain reaction (rT-qPcr) and western blotting were used to detect the expression levels of associated genes and proteins. iGF-1 (100 ng/ml) or VeGF (25 ng/ml) alone were revealed to be able to promote proliferation and migration of hcdPScs; however, the combined use of iGF-1 and VeGF enhanced this effect when compared with the use of either agent in isolation. alizarin red and alP staining revealed that the use of either VeGF or iGF-1 alone did not result in any significant effects, whereas their use in combination promoted the osteogenic differentiation of hcdPScs. in addition, the rT-qPcr and western blotting analyses revealed that the expression levels of runt-related transcription factor 2 (runX2), bone sialoprotein (BSP) and alP were increased upon combined treatment of the cells with VeGF and iGF-1. The expression levels of VeGF and plateletderived growth factor (PdGF) in hcdPScs were enhanced upon treatment with either VeGF or iGF-1 in isolation, with greater effects observed when VeGF and iGF-1 were added in combination, indicating that VeGF and iGF-1 may exert a synergistic role in these events. Further experiments revealed that the combination of VeGF and iGF-1 led to an activation of the aKT signaling pathway. The proliferation and angiogenesis of hcdPScs were also shown to be more effective compared with treatment with either VeGF or iGF-1 in isolation. Taken together, the present study has demonstrated that the combined use of VeGF and iGF-1 leads to an increase in the proliferation, migration, osteogenesis and angiogenesis of hcdPScs and, furthermore, these signaling molecules may mediate their effects via activation of the aKT signaling pathway.