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Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells.

ABSTRACT: BACKGROUND:Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. METHODS:Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. RESULTS:Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers' upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. CONCLUSIONS:The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs' osteogenic differentiation.

SUBMITTER: Wang Z

PROVIDER: S-EPMC6958786 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells.

Wang Zhuo Z Huang Yuanliang Y Tan Luanjun L

BMC developmental biology 20200114 1

<h4>Background</h4>Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs.<h4>Methods</h4>Microarray was used t ...[more]

PMID: 31931700

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Project description:BackgroundLong noncoding RNAs (lncRNAs) are regulatory molecules that participate in biological processes such as stem cell differentiation. Periodontal ligament stem cells (PDLSCs) exhibit great potential for the regeneration of periodontal tissue and the formation of new bone. However, although several lncRNAs have been found to be involved in the osteogenic differentiation of PDLSCs, the temporal transcriptomic landscapes of mRNAs and lncRNAs need to be mapped to obtain a complete picture of osteoblast differentiation. In this study, we aimed to characterize the time-course expression patterns of lncRNAs during the osteogenic differentiation of PDLSCs and to identify the lncRNAs that are related to osteoblastic differentiation.MethodsWe cultured PDLSCs in an osteogenic medium for 3, 7, or 14 days. We then used RNA sequencing (RNA-seq) to analyze the expression of the coding and non-coding transcripts in the PDLSCs during osteogenic differentiation. We also utilized short time-series expression miner (STEM) to describe the temporal patterns of the mRNAs and lncRNAs. We then performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to assess the biological relevance of genes in each profile, and used quantitative real-time PCR (qRT-PCR) to validate the differentially expressed mRNAs and lncRNAs that were associated with osteoblast differentiation. Lastly, we performed a knock down of two lncRNAs, MEG8, and MIR22HG, and evaluated the expression of osteogenic markers.ResultsWhen PDLSCs were differentiated to osteoblasts, mRNAs associated with bone remodeling, cell differentiation, and cell apoptosis were upregulated while genes associated with cell proliferation were downregulated. lncRNAs showed stage-specific expression, and more than 200 lncRNAs were differentially expressed between the undifferentiated and osteogenically differentiated PDLSCs. Using STEM, we identified 25 temporal gene expression profiles, among which 14 mRNA and eight lncRNA profiles were statistically significant. We found that genes in pattern 12 were associated with osteoblast differentiation. The expression patterns of osteogenic mRNAs (COL6A1, VCAN, RRBP1, and CREB3L1) and lncRNAs (MEG8 and MIR22HG) were consistent between the qRT-PCR and RNA-seq results. Moreover, the knockdown of MEG8 and MIR22HG significantly decreased the expression of osteogenic markers (runt-related transcription factor 2 and osteocalcin).DiscussionDuring the osteogenic differentiation of PDLSCs, both mRNAs and lncRNAs showed stage-specific expression. lncRNAs MEG8 and MIR22HG showed a high correlation with osteoblastogenesis. Our results can be used to gain a more comprehensive understanding of the molecular events regulating osteoblast differentiation and the identification of functional lncRNAs in PDLSCs.

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Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells.

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Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells.

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