LncRNA H19 facilitates the proliferation and differentiation of human dental pulp stem cells via EZH2-dependent LATS1 methylation.
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ABSTRACT:
SUBMITTER: Du Z
PROVIDER: S-EPMC8339349 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Increasing evidence has revealed that long non-coding RNAs (lncRNAs) exert critical roles in biological mineralization. As a critical process for dentin formation, odontoblastic differentiation is regulated by complex signaling networks. The present study aimed to investigate the biological role and regulatory mechanisms of lncRNA-H19 (H19) in regulating the odontoblastic differentiation of human dental pulp stem cells (hDPSCs). METHODS:We performed lncRNA microarray assay to reveal the expression patterns of lncRNAs involved in odontoblastic differentiation. H19 was identified and verified as a critical factor by qRT-PCR. The gain- and loss-of-function studies were performed to investigate the biological role of H19 in regulating odontoblastic differentiation of hDPSCs in vitro and in vivo. Odontoblastic differentiation was evaluated through qRT-PCR, Western blot, and Alizarin Red S staining. Bioinformatics analysis identified that H19 could directly interact with miR-140-5p, which was further verified by luciferase reporter assay. After overexpression of miR-140-5p in hDPSCs, odontoblastic differentiation was determined. Moreover, the potential target genes of miR-140-5p were investigated and the biological functions of BMP-2 and FGF9 in hDPSCs were verified. Co-transfection experiments were conducted to validate miR-140-5p was involved in H19-mediated odontoblastic differentiation in hDPSCs. RESULTS:The expression of H19 was significantly upregulated in hDPSCs undergoing odontoblastic differentiation. Overexpression of H19 stimulated odontoblastic differentiation in vitro and in vivo, whereas downregulation of H19 revealed the opposite effect. H19 binds directly to miR-140-5p and overexpression of miR-140-5p inhibited odontoblastic differentiation of hDPSCs. H19 acted as a miR-140-5p sponge, resulting in regulated the expression of BMP-2 and FGF9. Overexpression of H19 abrogated the inhibitory effect of miR-140-5p on odontoblastic differentiation. CONCLUSION:Our data revealed that H19 plays a positive regulatory role in odontoblastic differentiation of hDPSCs through miR-140-5p/BMP-2/FGF9 axis, suggesting that H19 may be a stimulatory regulator of odontogenesis.
| S-EPMC7251819 | biostudies-literature
Project description:Differentiation potency of human dental pulp cells (hDPCs) is essential for dentin regeneration. DNA methylation is one of the major epigenetic mechanisms and is suggested to involve in differentiation of hDPCs, the machinery of which includes DNA methyltransferase enzymes (DNMTs) and methyl-CpG-binding domain proteins (MBDs). Our previous study has found that melatonin (MT) promoted hDPC differentiation, but its mechanism remains elusive. We aimed to investigate the role of DNA methylation in the promotion of MT to differentiation of hDPCs in vitro. hDPCs were cultured in basal growth medium (CO) or odontogenic medium (OM) exposed to MT at different concentrations (0, 10-12, 10-10, 10-8, 10-6, 10-4 M). The cell growth was analyzed using Cell Counting Kit-8 assay, and mineralized tissue formation was measured using Alizarin red staining. The expression of the 10 genes (DNMT1, DNMT3A, DNMT3B, MBD1-6, MeCP2) was determined using real-time qPCR and western blotting. The abundance of MeCP2 in the nuclei was evaluated using immunofluorescence analysis. Global methylation level was tested using ELISA. We found that mineralized tissue formation significantly increased in OM with MT at 10-4 M, while the levels of MeCP2 and global DNA methylation level declined. The expression of MBD1, MBD3, and MBD4 significantly increased in OM alone, and the expession of DNMT1 and MBD2 was decreased. These results indicate that MT promotes odontogenic differentiation of hDPCs in vitro by regulating the levels of DNMT1, MeCP2, and global DNA methylation, suggesting that MT-induced DNA methylation machinery may play an important role in tooth regeneration.
| S-EPMC8291816 | biostudies-literature
Project description:Aim:The purpose of the current study was to investigate the effects of nephronectin (Npnt) in human dental pulp stem cells (hDPSCs). Methodology:Npnt was coated to nontissue culture-treated polystyrene (non-PS) plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-), fish scale type I collagen- (FCOL1-), and human fibronectin- (Fn-) coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively. Results:Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD) motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs. Conclusions:The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.
| S-EPMC5735320 | biostudies-literature
Project description:Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
| S-EPMC3875457 | biostudies-literature
Project description:Small non-coding microRNAs (miRNAs) are known to play crucial roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In an attempt to search for novel miRNAs that can control the fate of dental tissue-derived stem cells, we sorted side population (SP) cells, known to be enriched in stem cells, from dental pulp cells (DPCs) and periodontal ligament cells (PDLCs), and performed a miRNA array. As a result, miR-200b and miR-607 were highly expressed in SP cells, whereas miR-1260b and miR-720 was highly expressed in the differentiated main population (MP) cells. Of note, gain-and-loss of function analysis showed that miR-720 regulates the expression of pluripotency factor NANOG and DNA methyltransferases DNMT3A, DNMT3B and DNMT1 in DPCs. Knockdown of miR-720 significantly increased the levels of NANOG as well as the number of cells positive to the stem cell marker SSEA-4, but down-regulated the levels of DNMTs. miR-720 also regulated the proliferation of DPCs as determined by immunocytochemical analysis against ki-67, as well as odontogenic differentiation demonstrated by alizarin red staining, alkaline phosphatase activity and osteopontin mRNA level. Our findings identify mi-720 as a novel miRNA involved in the regulation of stem cell fate of DPCs. Two cell types (dental pulp cells(DPCs), periodontal ligament cells(PDLCs)) were sorted by FACS to isolate side population (SP) and main population (MP) cells. Then a microRNA array was performed with the SP and MP cells of DPCs and PDLCs.
2013-05-17 | E-GEOD-47025 | biostudies-arrayexpress
Project description:Dental pulp stem cells (DPSCs), which can differentiate into several types of cells, are subjected to mechanical stress by jaw movement and occlusal forces. In this study, we evaluated how the uniaxial mechanical stretch influences proliferation and differentiation of DPSCs. DPSCs were isolated and cultured from male Sprague-Dawley rats. Cultured DPSCs were identified by surface markers and the differentiation capabilities as adipocytes or osteoblasts. To examine the response to mechanical stress, uniaxial stretch was exposed to cultured DPSCs. We evaluated the impact of stretch on the intracellular signaling, proliferation, osteogenic differentiation, and gene expressions of DPSCs. Stretch increased the phosphorylation of Akt, ERK1/2, and p38 MAP kinase as well as the proliferation of DPSCs. The stretch-induced proliferation of DPSCs was abolished by the inhibition of the ERK pathway. On the other hand, stretch significantly decreased the osteogenic differentiation of DPSCs, but did not affect the adipogenic differentiation. We also confirmed mRNA expressions of osteocalcin and osteopontin were significantly suppressed by stretch. In conclusion, uniaxial stretch increased the proliferation of DPSCs, while suppressing osteogenic differentiation. These results suggest a crucial role of mechanical stretch in the preservation of DPSCs in dentin. Furthermore, mechanical stretch may be a useful tool for increasing the quantity of DPSCs in vitro for regenerative medicine.
| S-EPMC3566654 | biostudies-literature
Project description:Mesenchymal stem cells are a highly promising source of cells for regeneration therapy because of their multilineage differentiation potential. However, distinct markers for mesenchymal stem cells are not well-established. To identify new candidate marker genes for multipotent human dental pulp stem cells, we analyzed the characteristics and gene expression profiles of cell clones obtained from a single dental pulp specimen derived from an 11-year-old female patient. Fifty colony-forming single cell-derived clones were separately cultured until the cessation of growth. These clones varied in their proliferation abilities and surface marker (STRO-1 and CD146) expression patterns, as well as their odontogenic, adipogenic, and chondrogenic differentiation potentials. Four clones maintained their original differentiation potentials during long-term culture. Gene expression profile by DNA microarray analysis of five representative clones identified 1227 genes that were related to multipotency. Ninety of these 1227 genes overlapped with genes reportedly involved in 'stemness or differentiation'. Based on the predicted locations of expressed protein products and large changes in expression levels, 14 of the 90 genes were selected as candidate dental pulp stem cell markers, particularly in relation to their multipotency characteristics. This characterization of cell clones obtained from a single specimen of human dental pulp provided information regarding new candidate marker genes for multipotent dental pulp stem cells, which could facilitate efficient analysis or enrichment of multipotent stem cells.
| S-EPMC7324427 | biostudies-literature
Project description:Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
2013-05-17 | GSE47025 | GEO
Project description:Objective To investigate the role of lncRNA-BLACAT1 in promoting H3K27 trimethylation of CDKN1C gene by recruiting EZH2 to regulate CCNE on glycolysis and mitochondrial oxidative phosphorylation of pancreatic cancer (PC) cells. Methods Following bioinformatic prediction, EZH2 and BLACAT1 in PC cells were interfered, and cells proliferation, migration and invasion in each group were detected. Western blotting detected the expression of key proteins of mitochondrial complex. The sub-cellular localization of BLACAT1 was tested, followed by testing the binding of CDKN1C and BLACAT1 with EZH2, followed by in vivo verification. Results Based on bioinformatic prediction, EZH2 and BLACAT1 were highly expressed in PC, while CDKN1C was lowly expressed (all P < 0.05). Interference with EZH2 and BLACAT1 inhibited cell proliferation, migration and aerobic glycolysis, and promoted mitochondrial oxidative phosphorylation (all P < 0.05). BLACAT1 promoted H3K27 trimethylation of CDKN1C through recruiting EZH2 (all P < 0.05). In vivo results showed that BLACAT1 interference inhibited tumor formation (all P < 0.05). Conclusion Interference with BLACAT1 inhibits H3K27 trimethylation of CDKN1C gene by blocking EZH2 recruitment to promote CDKN1C expression and inhibit CCNE expression, thus suppressing PC cell proliferation, migration and aerobic glycolysis, and promoting mitochondrial oxidative phosphorylation.
| S-EPMC7538708 | biostudies-literature
Project description:Dental pulp stem cells (DPSCs) are a unique precursor population isolated from postnatal human dental pulp and have the ability to regenerate a reparative dentin-like complex. Canonical Wnt signaling plays a critical role in tooth development and stem cell self-renewal through beta-catenin. In this study, the regulation of odontoblast-like differentiation of DPSCs by canonical Wnt signaling was examined. DPSCs were stably transduced with canonical Wnt-1 or the active form of beta-catenin, with retrovirus-mediated infection. Northern blot analysis found that Wnt-1 strongly induced the expression of matricellular protein osteopontin, and modestly enhanced the expression of type I collagen in DPSCs. Unexpectedly, Wnt-1 inhibited alkaline phosphatase (ALP) activity and the formation of mineralized nodules in DPSCs. Moreover, over-expression of beta-catenin was also sufficient to suppress the differentiation and mineralization of DPSCs. In conclusion, our results suggest that canonical Wnt signaling negatively regulates the odontoblast-like differentiation of DPSCs.
| S-EPMC2906770 | biostudies-literature