Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway
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Open Access
- 31 August 2018
- journal article
- research article
- Published by Springer Science and Business Media LLC in Stem Cell Research & Therapy
- Vol. 9 (1), 1-14
- https://doi.org/10.1186/s13287-018-0976-0
Abstract
Periodontal 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. The 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. We 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. Our 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.Keywords
Funding Information
- National Natural Science Foundation of China (81670957, 81772876, 81700938)
- Beijing Municipal Natural Science Foundation (7172239)
- the sponsored by the Fund for Fostering Young Scholars of Peking University Health Science Center (BMU2018PY022)
This publication has 50 references indexed in Scilit:
- Osteogenic Differentiation Capacity of Human Skeletal Muscle-Derived Progenitor CellsPLOS ONE, 2013
- Nectandrin A Enhances the BMP-Induced Osteoblastic Differentiation and Mineralization by Activation of p38 MAPK-Smad Signaling PathwayKorean Journal of Physiology & Pharmacology, 2013
- Circular RNAs are abundant, conserved, and associated with ALU repeatsRNA, 2012
- Transcriptome-wide discovery of circular RNAs in ArchaeaNucleic Acids Research, 2011
- miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNAThe EMBO Journal, 2011
- Testing the Critical Size in Calvarial Bone Defects: Revisiting the Concept of a Critical-Size DefectPlastic and Reconstructive Surgery, 2010
- Receptor oligomerization and beyond: a case study in bone morphogenetic proteinsBMC Biology, 2009
- Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cellsArthritis Research & Therapy, 2009
- A Single Residue of GDF-5 Defines Binding Specificity to BMP Receptor IBJournal of Molecular Biology, 2005
- The periodontal ligament: a unique, multifunctional connective tissuePeriodontology 2000, 1997