Dataset Information

MiR-140-5p regulates the odontoblastic differentiation of dental pulp stem cells via the Wnt1/?-catenin signaling pathway.

ABSTRACT:

Background

MicroRNAs (miRNAs) play a key role in regulating cell differentiation. In the present study, we aimed to explore the role of miR-140-5p in odontoblastic differentiation of dental pulp stem cells (DPSCs).

Methods

DPSCs from normal human impacted third molars were isolated and cultured. After overexpression or silencing of miR-140-5p in DPSCs, activity, proliferation, and odontoblastic differentiation of DPSCs were evaluated. The possible target gene of miR-140-5p was verified by luciferase reporter gene assay. Using gene transfection technology, RT-CPR, and Western blot to confirm miR-140-5p regulates the odontoblastic differentiation of DPSCs through Wnt1/?-catenin signaling.

Results

We found the expression of miR-140-5p decreased in the differentiated DPSCs for odontoblastic cells, and at the same time, the expressions of Wnt1 and ?-catenin increased. Wnt1 was the target gene of miR-140-5p which was confirmed by luciferase reporter gene system. miR-140-5p overexpression suppressed the expression of Wnt1. miR-140-5p inhibitor could promote the odontoblastic differentiation of DPSCs. miR-140-5p mimic could weaken the odontoblastic differentiation of DPSCs, which could be reversed by the overexpression of Wnt1.

Conclusion

Our data demonstrated that miR-140-5p regulates the odontoblastic differentiation of DPSCs via targeting Wnt1/?-catenin signaling. Therefore, miR-140-5p might be a molecular target to regulate the odontoblastic differentiation for the therapeutic agents in dental medicine.

SUBMITTER: Lu X

PROVIDER: S-EPMC6664499 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

miR-140-5p regulates the odontoblastic differentiation of dental pulp stem cells via the Wnt1/β-catenin signaling pathway.

Lu Xiaohui X Chen Xi X Xing Jing J Lian Min M Huang Dan D Lu Yuanzhou Y Feng Guijuan G Feng Xingmei X

Stem cell research & therapy 20190729 1

<h4>Background</h4>MicroRNAs (miRNAs) play a key role in regulating cell differentiation. In the present study, we aimed to explore the role of miR-140-5p in odontoblastic differentiation of dental pulp stem cells (DPSCs).<h4>Methods</h4>DPSCs from normal human impacted third molars were isolated and cultured. After overexpression or silencing of miR-140-5p in DPSCs, activity, proliferation, and odontoblastic differentiation of DPSCs were evaluated. The possible target gene of miR-140-5p was ver ...[more]

PMID: 31358066

Similar Datasets

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.

Project description:Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Project description:Although the cancer/testis antigen CAGE has been implicated in tumorigenesis, the molecular mechanisms of CAGE-promoted tumorigenesis remain largely unknown. CT26Flag-CAGE cells, CT26 (mouse colon cancer cells) cells stably expressing CAGE, were established to investigate CAGE-promoted tumorigenesis. Down-regulation of CAGE led to decreased autophagic flux in CT26Flag-CAGE cells. CAGE interacted with Beclin1, a mediator of autophagy. The CT26Flag-CAGE cells showed enhanced autophagosome formation and displayed greater tumor spheroid-forming potential than CT26 cells. MicroRNA array analysis revealed that CAGE decreased the expression of various microRNAs, including miR-140-5p, in CT26 cells. CAGE was shown to bind to the promoter sequences of miR-140-5p. MiR-140-5p inhibition increased the tumorigenic potential of and autophagic flux in CT26 cells. A miR-140-5p mimic exerted negative effects on the tumorigenic potential of CT26Flag-CAGE cells and autophagic flux in CT26Flag-CAGE cells. MiR-140-5p was predicted to bind to the 3'-UTR of Wnt1. CT26Flag-CAGE cells showed higher expression of Wnt1 than CT26 cells. Down-regulation of Wnt1 decreased autophagic flux. Luciferase activity assays showed the direct regulation of wnt1 by miR-140-5p. Tumor tissue derived from the CT26Flag-CAGE cells revealed higher expressions of factors associated with activated mast cells and tumor-associated macrophages than tumor tissue derived from CT26 cells. Culture medium from the CT26Flag-CAGE cells increased autophagic flux in CT26 cells, mast cells and macrophages. Culture medium from the CT26Flag-CAGE cells increased CD163 and autophagic flux in CT26 cells, mast cells, and macrophages in a Wnt1-dependent manner. Exosomes from CT26Flag-CAGE cells increased autophagc flux in CT26 cells, mast cells, and macrophages. Exosomes from CT26Flag-CAGE cells increased the tumorigenic potential of CT26 cells. Wnt1 was shown to be present within the exosomes. Recombinant Wnt1 protein increased autophagic flux in CT26, mast cells, and macrophages. Recombinant wnt1 protein mediated interactions between the CT26 cells, mast cells, and macrophages. Our results showed novel roles for the CAGE-miR-140-5p-Wnt1 axis in autophagic flux and cellular interactions mediated by exosomes.