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Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature.

ABSTRACT: Idiopathic short stature (ISS) is a main reason for low height among children. Its exact aetiology remains unclear. Recent findings have suggested that the aberrant expression of circRNAs in peripheral blood samples is associated with many diseases. However, to date, the role of aberrant circRNA expression in mediating ISS pathogenesis remains largely unknown. The up-regulated circANAPC2 was identified by circRNA microarray analysis and RT-qPCR. Overexpression of circANAPC2 inhibited the proliferation of human chondrocytes, and cell cycle was arrested in G1 phase. The expressions of collagen type X, RUNX2, OCN and OPN were significantly down-regulated following circANAPC2 overexpression. Moreover, Von Kossa staining intensity and alkaline phosphatase activity were also decreased. Luciferase reporter assay results showed that circANAPC2 could be targeted by miR-874-3p. CircANAPC2 overexpression in human chondrocytes inhibits the expression of miR-874-3p. The co-localization of circANAPC2 and miR-874-3p was confirmed in both human chondrocytes and murine femoral growth plates via in situ hybridization. The rescue experiment demonstrated that the high expression of miR-874-3p overexpression antagonized the suppression of endochondral ossification, hypertrophy and chondrocyte growth caused by circANAPC2 overexpression. A high-throughput screening of mRNA expression and RT-qPCR verified SMAD3 demonstrated the highest different expressions following overcircANAPC2. Luciferase reporter assay results indicated that miR-874-3p could be targeted by Smad3, thus down-regulating the expression of Smad3. Subsequent rescue experiments of SMAD3 further confirmed that circANAPC2 suppresses endochondral ossification, hypertrophy and chondrocyte growth through miR-874-3p/Smad3 axis. The present study provides evidence that circANAPC2 can serve as a promising target for ISS treatment.

SUBMITTER: Liu X

PROVIDER: S-EPMC8034469 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Circular (circ)RNAs are an important group of non‑coding RNAs involved in different pathological and physiological functions, such as longitudinal bone growth. However, the effects of an increase or decrease in circRNA expression on idiopathic short stature (ISS) remain largely unknown. The present study compared the circRNA expression patterns of patients with ISS and healthy individuals to identify differentially expressed circRNAs involved in the regulation of ISS pathogenesis and their target microRNAs (miR). Microarray analysis revealed that 145 circRNAs were differentially expressed in patients with ISS, including 83 up‑ and 62 downregulated circRNAs. Reverse transcription‑quantitative PCR confirmed that hsa_circRNA_0079201 was increased in patients with ISS compared with that in the normal individuals, whilst hsa_circRNA_0079201 overexpression in human chondrocytes was shown to significantly suppress their proliferation, hypertrophy and endochondral ossification abilities. Luciferase reporter assays identified that circRNA_0079201 acted as an miR‑140‑3p sponge. In situ hybridization confirmed the co‑localization of circRNA_0079201 and miR‑140‑3p in the human chondrocyte and neonatal femur growth plate of C57 mice, while rescue experiments demonstrated that miR‑140‑3p overexpression reversed the inhibition of human chondrocyte proliferation, hypertrophy and endochondral ossification, caused by circRNA_0079201 overexpression. Bioinformatics analysis and luciferase reporter assays revealed that SMAD2 was a potential target gene of miR‑140‑3p. Furthermore, overexpressing circRNA_0079201 in human chondrocytes suppressed miR‑140‑3p and increased SMAD2 protein expression level. Taken together, chondrocyte proliferation, hypertrophy and endochondral ossification in ISS was suppressed by a novel regulatory axis consisting of the hsa_circRNA_0079201/miR‑140‑3p/SMAD2 pathway. The present study provided evidence that hsa_circRNA_0079201 may be a potential target for ISS therapy.

Project description:The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Project description:Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of ?-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14?days-of-age (n?=?5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9?days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14?days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14?days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/?-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.

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Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR-874-3p/SMAD3 signalling pathway in idiopathic short stature.

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