Dataset Information

Long Noncoding RNA GAS5 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating GDF5 and p38/JNK Signaling Pathway.

ABSTRACT: Both extracellular matrix (ECM) and stem cells contribute to the formation of bones. Accumulating evidence proved that the growth differentiation factor 5 (GDF5) plays a vital role in ECM osteogenesis regulation; the use of human periodontal ligament stem cells (hPDLSCs) may contribute to alveolar bone regeneration. Moreover, long noncoding RNAs (lncRNA) serves as a regulator in the growing process of cellular organisms including bone formation. Previous efforts has led us to the discovery that the expression of growth arrest specific transcript 5 (GAS5) changed in the osteogenic differentiation of hPDLSCs. Moreover, the expression of GAS5, as it turns out, is correlated to GDF5. This study attempts to investigate the inner workings of GAS5 in its regulation of osteoblastic differentiation of hPDLSCs. Cell transfection, Alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, qRT-PCR, immunofluorescence staining analysis and western blotting were employed in this study. It came to our notice that GAS5 and GDF5 expression increased during osteogenesis induction of hPDLSCs. Knocking down of GAS5 inhibited the osteogenic differentiation of hPDLSCs, whereas overexpressing GAS5 promoted these effects. Molecular mechanism study further demonstrated that overexpressing GAS5 bolsters GDF5 expression and boosts the phosphorylation of JNK and p38 in hPDLSCs, with opposite effects in GAS5 knockdown group. To sum up, long noncoding RNA GAS5 serves to regulate the osteogenic differentiation of PDLSCs via GDF5 and p38/JNK signaling pathway. Our findings expand the theoretical understanding of the osteogenesis mechanism in hPDLSCs, providing new insights into the treatment of bone defects.

SUBMITTER: Yang Q

PROVIDER: S-EPMC7251029 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Long Noncoding RNA GAS5 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating GDF5 and p38/JNK Signaling Pathway.

Yang Qiaolin Q Han Yineng Y Liu Peng P Huang Yiping Y Li Xiaobei X Jia Lingfei L Zheng Yunfei Y Li Weiran W

Frontiers in pharmacology 20200520

Both extracellular matrix (ECM) and stem cells contribute to the formation of bones. Accumulating evidence proved that the growth differentiation factor 5 (GDF5) plays a vital role in ECM osteogenesis regulation; the use of human periodontal ligament stem cells (hPDLSCs) may contribute to alveolar bone regeneration. Moreover, long noncoding RNAs (lncRNA) serves as a regulator in the growing process of cellular organisms including bone formation. Previous efforts has led us to the discovery that ...[more]

PMID: 32508644

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Project description:BackgroundPeriodontal ligament stem cells (PDLSCs) are considered as candidate cells for the regeneration of periodontal and alveolar bone tissues. Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), which is a newly discovered circular RNA (circRNA), has been reported to act as an miR-7 sponge and to be involved in many biological processes. Here, we investigated the potential roles of CDR1as and miR-7 in the osteogenic differentiation of PDLSCs.MethodsThe expression pattern of CDR1as and miR-7 in PDLSCs during osteogenesis was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Then we overexpressed or knocked down CDR1as or miR-7 to confirm whether they were involved in the regulation of osteoblast differentiation in PDLSCs. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were used to detect the activity of osteoblasts and mineral deposition. Furthermore, a dual luciferase reporter assay was conducted to analyze the binding of miR-7 to growth differentiation factor (GDF)5. To further verify the role of CDR1as in osteoblast differentiation, we conducted animal experiments in vivo. New bone formation in specimens was analyzed by microcomputed tomography (micro-CT), hematoxylin and eosin staining, and immunofluorescence staining.ResultsWe observed that CDR1as was significantly upregulated during the osteogenic differentiation, whereas miR-7 was significantly downregulated. Moreover, knockdown of CDR1as and overexpression of miR-7 inhibited the ALP activity, ARS staining, and expression of osteogenic genes. Overexpression of miR-7 significantly reduced the activity of luciferase reporter vectors containing the wild-type, but not the mutant, 3' untranslated region (UTR) sequence of GDF5. Furthermore, knockdown of GDF5 partially reversed the effects of miR-7 inhibitor on osteoblast differentiation. Downregulation of CDR1as or GDF5 subsequently inhibited phosphorylation of Smad1/5/8 and p38 mitogen-activated protein kinases (MAPK), while upregulation of miR-7 decreased the level of phosphorylated Smad1/5/8 and p38 MAPK. In vivo, CDR1as knockdown lead to less bone formation compared with the control group as revealed by micro-CT and the histological analysis.ConclusionsOur results demonstrated that CDR1as acts as a miR-7 inhibitor, triggering the upregulation of GDF5 and subsequent Smad1/5/8 and p38 MAPK phosphorylation to promote osteogenic differentiation of PDLSCs. This study provides a novel understanding of the mechanisms of osteogenic differentiation, and suggests a potential method for promoting bone formation.

Project description: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.

Dataset Information

Long Noncoding RNA GAS5 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating GDF5 and p38/JNK Signaling Pathway.

Publications

Long Noncoding RNA GAS5 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating GDF5 and p38/JNK Signaling Pathway.

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