Dataset Information

MiR-497-5p Regulates Osteo/Odontogenic Differentiation of Stem Cells From Apical Papilla via the Smad Signaling Pathway by Targeting Smurf2.

ABSTRACT: Osteo/odontogenic differentiation is a key process of human stem cells from apical papilla (SCAP) in tooth root development. Emerging evidence indicates microRNAs (miRNAs) play diverse roles in osteogenesis. However, their functions in osteo/odontogenic differentiation of SCAP require further elucidation. To investigate the role of miRNA in SCAP osteo/odontogenic differentiation and underlying mechanisms, miRNA microarray analysis was performed to screen differentially expressed miRNAs between control and osteo/odontogenic-induced group. Quantitative real-time PCR (qRT-PCR) and western blot were used to detected osteo/odontogenic differentiation-related markers and possible signaling pathway SCAP-associated genes. Alizarin Red Staining (ARS) were applied to evaluated osteogenic capacity. The results showed that miR-497-5p increased during SCAP osteo/odontogenic differentiation. Overexpression of miR-497-5p enhanced the osteo/odontogenic differentiation of SCAP, whereas downregulation of miR-497-5p elicited the opposite effect, thus suggesting that miR-497-5p is a positive regulator of the osteo/odontogenic differentiation of SCAP. Bioinformatic analysis and dual luciferase reporter assay identified that SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) is a direct target of miR-497-5p. Further study demonstrated that Smurf2 negatively regulates SCAP osteo/odontogenic differentiation, and silencing Smurf2 could block the inhibitory effect of the miR-497-5p inhibitor. Meanwhile, pathway detection manifested that miR-497-5p promotes osteo/odontogenic differentiation via Smad signaling pathway. Collectively, our findings demostrate that miR-497-5p promotes osteo/odontogenic differentiation of SCAP via Smad signaling pathway by targeting Smurf2.

SUBMITTER: Liu J

PROVIDER: S-EPMC7662069 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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MiR-497-5p Regulates Osteo/Odontogenic Differentiation of Stem Cells From Apical Papilla via the Smad Signaling Pathway by Targeting <i>Smurf2</i>.

Liu Junqing J Wang Xiaolong X Song Mengxiao M Du Jing J Yu Jiali J Zheng Wenzhou W Zhang Chengfei C Wang Yan Y

Frontiers in genetics 20201030

Osteo/odontogenic differentiation is a key process of human stem cells from apical papilla (SCAP) in tooth root development. Emerging evidence indicates microRNAs (miRNAs) play diverse roles in osteogenesis. However, their functions in osteo/odontogenic differentiation of SCAP require further elucidation. To investigate the role of miRNA in SCAP osteo/odontogenic differentiation and underlying mechanisms, miRNA microarray analysis was performed to screen differentially expressed miRNAs between c ...[more]

PMID: 33193708

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Project description:Stem cells from apical papilla (SCAP) possess clear osteo?/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo?/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo?/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector?mediated gene transfection was conducted with SCAP in order to construct blank vector?transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene?transfected SCAP (SCAP?VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP?VEGF was demonstrated to exhibit increased proliferation, and SCAP?BMP2?VEGF exhibited reduced proliferation during eight days observation. SCAP?BMP2?VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP?BMP2?VEGF group exhibited a significantly greater number of ALP?positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo?/odontogenic differentiation of human SCAP.

Project description:IntroductionPeriapical alveolar bone loss is the common consequence of apical periodontitis (AP) caused by persistent local inflammation around the apical area. Human stem cells from apical papilla (hSCAPs) play a crucial role in the restoration of bone lesions during AP. Studies have recently identified the critical role of microRNAs (miRNAs) involved in AP pathogenesis, but little is known about their function and potential molecular mechanism, especially in the osteogenesis of hSCAPs during AP. Here, we investigated the role of clinical sample-based specific miRNAs in the osteogenesis of hSCAPs.MethodsDifferential expression of miRNAs were detected in the periapical tissues of normal and patients with AP via transcriptomic analysis, and the expression of miR-199a-5p was confirmed by qRT-PCR. Treatment of hSCAPs with miR-199a-5p mimics while loaded onto beta-tricalcium phosphate (β-TCP) ceramic particle scaffold to explore its effect on osteogenesis in vivo. RNA binding protein immunoprecipitation (RIP) and Luciferase reporter assay were conducted to identify the target gene of miR-199a-5p.ResultsThe expression of miR-199a-5p was decreased in the periapical tissues of AP patients, and miR-199a-5p mimics markedly enhanced cell proliferation and osteogenic differentiation of hSCAPs, while miR-199a-5p antagomir dramatically attenuated hSCAPs osteogenesis. Moreover, we identified and confirmed Interferon Induced Protein with Tetratricopeptide Repeats 2 (IFIT2) as a specific target of miR-199a-5p, and silencing endogenous IFIT2 expression alleviated the inhibitory effect of miR-199a-5p antagomir on the osteogenic differentiation of hSCAPs. Furthermore, miR-199a-5p mimics transfected hSCAPs loaded onto beta-tricalcium phosphate (β-TCP) scaffolds induced robust subcutaneous ectopic bone formation in vivo.DiscussionThese results strengthen our understanding of predictors and facilitators of the key AP miRNAs (miR-199a-5p) in bone lesion repair under periapical inflammatory conditions. And the regulatory networks will be instrumental in exploring the underlying mechanisms of AP and lay the foundation for future regenerative medicine based on dental mesenchymal stem cells.

Project description:BackgroundThe differentiation of stem cells from exfoliated deciduous teeth (SHEDs) into odontoblasts determines the regeneration of dentin-pulp complex. Non-coding RNAs (ncRNAs), including microRNA (miRNA) and long non-coding RNA (lncRNA), participate in many multiple biological processes, but the specific miRNAs involved in odontogenesis are incompletely defined. It was confirmed that lncRNA IGFBP7-AS1 could positively regulate odontogenetic differentiation in SHEDs. To investigate the downstream mechanisms of this process, miR-335-3p and miR-155-5p were found to be closely related with SHED odontogenic differentiation through whole-genome sequencing. The aim of the current study was to determine the role of miR-335-3p/miR-155-5p in IGFBP7-AS1-enhanced SHED differentiation and explore the potential mechanism of IGFBP7-AS1-mediated odontogenesis.MethodsPutative miR-335-3p/miR-155-5p binding sites within IGFBP7-AS1 were identified by bioinformatics analysis, and the binding of miR-335-3p/miR-155-5p to these sites was confirmed by dual-luciferase reporter gene assays. The effects of miR-335-3p/miR-155-5p in odontogenesis were detected by tissue nonspecific alkaline phosphatase staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR) analyses, and western blot testing. The molecular mechanisms of miR-335-3p/miR-155-5p involved in IGFBP7-AS1-mediated odontogenesis were analysed by qRT-PCR and western blot testing.ResultsDual-luciferase reporter gene assays showed that miR-335-3p/miR-155-5p could directly bind to IGFBP7-AS1. MiR-335-3p and miR-155-5p both could down-regulate dentin sialophosphoprotein expression, and both miRNAs could inhibit IGFBP7-AS1-mediated SHED odontogenetic differentiation via suppression of the extracellular signal-regulated kinase (ERK) pathway.ConclusionsBoth miR-335-3p and miR-155-5p were negative regulators to IGFBP7-AS1-enhanced odontogenic differentiation of SHED through suppression of the ERK pathway.

Dataset Information

MiR-497-5p Regulates Osteo/Odontogenic Differentiation of Stem Cells From Apical Papilla via the Smad Signaling Pathway by Targeting Smurf2.

Publications

MiR-497-5p Regulates Osteo/Odontogenic Differentiation of Stem Cells From Apical Papilla via the Smad Signaling Pathway by Targeting <i>Smurf2</i>.

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