Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0976-0) contains supplementary material, which is available to authorized users.

hsa_circRNA_100533 regulates GNAS by sponging hsa_miR_933 to prevent oral squamous cell carcinoma

“…It was showed that OSCC tissues have abundant circRNAs, and multiple circRNAs significantly differentially expressed. It was reported that hsa_circ_0008309 and hsa_circ_001242 was significantly downregulated in OSCC tissues, correlated with pathological differentiation of OSCC, and may act as a potential novel biomarker for the diagnosis and treatment of OSCC . In addition, salivary hsa_circ_0001874 and hsa_circ_0001971 also act as biomarkers for diagnosing OSCC .…”

“…miR‐182‐5p promoted tumor growth of OSCC through inhibition of CAMK2N1 . miR‐218‐5p was downregulated in OSCC and it promoted invasion of oral squamous cell carcinoma cells by activating CD44‐ROCK . Although miRNAs have been extensively studied in OSCC, there are still a large number of unknown miRNAs needed further study to discover the unknown functions and mechanisms.…”

“…Circular RNA (circRNA) is one kind of noncoding RNAs, characterized by a covalently closed loop, without a 5′cap or a 3′poly (A) tail, the structure of which account for its stability and resistance to RNA exonucleases. Increasing evidence showed that circRNAs are involved in various physiological and pathological processes, such as proliferation, apoptosis, invasion, differentiation, and aging . Recently, multiple circRNAs have been found to be the biomarkers of different diseases, especially cancers .…”

The roles of circRFWD2 and circINO80 during NELL‐1‐induced osteogenesis

“…Mm9_circ_009056 could act as a sponge of miR‐22‐3p, regulating the expression of BMP7 to promote bone formation . CircRNA CDR1as could down‐regulate miR‐7 to activate p38 MAPK pathway, which enhanced the osteogenesis of periodontal ligament stem cells . However, the roles of circRNAs in recombinant NELL‐1‐induced osteogenesis of hASCs remains unclear.…”

Altered expression of circular RNAs in human placental chorionic plate‐derived mesenchymal stem cells pretreated with hypoxia

“…CircRNA BANP and circRNA ITCH can interact with miRNA34a and miRNA146a to regulate osteogenic differentiation of the periodontal ligament stem cells via the MAPK pathway . Promotion of osteogenic differentiation of the periodontal ligament stem cells related to circRNA CDR1as is modulated through the Smad‐dependent and p38 MAPK signaling pathways …”