Circ-SPATA13 regulates the osteogenic differentiation of human periodontal ligament stem cells through the miR-485-5p_R + 1/BMP7 axis

Background

Human periodontal ligament stem cells (PDLSCs) are widely available and have strong osteogenic differentiation ability, which makes them promising tools for bone regeneration. Circular RNAs (circRNAs) play a variety of functions in the process of cell differentiation and are potential therapeutic targets. Here, we identified a new circRNA, circ-SPATA13, and found that it was highly positively correlated with the osteogenic differentiation of PDLSCs. Therefore, in this study, we revealed the significance and mechanism of circ-SPATA13 in the osteogenic differentiation of PDLSCs.

Methods

PDLSCs were isolated from third molars with incomplete apical development and induced to undergo chondrogenic, adipogenic, or osteogenic differentiation. Surface markers were detected via flow cytometry. Proliferation was assessed with EdU and CCK-8 assays. The circ-SPATA13 and miR-485-5p_R + 1-mediated control of mineral deposition was evaluated through alizarin red and alkaline phosphatase staining. Osteogenesis-related factor expression was detected through western blotting, immunofluorescence, and qRT-PCR. Fluorescence in situ hybridization was used to examine circ-SPATA13 localization within PDLSCs. The relationships among circ-SPATA13, miR-485-5p_R + 1, and BMP7 during PDLSCs osteogenesis were assessed through western blotting, qRT-PCR, dual-luciferase assay, rescue experiment, and bioinformatics approaches.

Results

Primary PDLSCs expressing mesenchymal stem cell surface markers were isolated. Circ-SPATA13 was identified and found to have no impact on PDLSC proliferation, whereas it was a positive regulator of their osteogenic differentiation, a process which was antagonized by miR-485-5p_R + 1. Dual-luciferase reporter assays revealed that circ-SPATA13 was able to function as a molecular sponge to sequester miR-485-5p_R + 1 within PDLSCs, while this miRNA was able to bind to the 3’-UTR of the target mRNA BMP7. In rescue experiments, circ-SPATA13 was confirmed to regulate the osteogenic differentiation of PDLSCs via this miR-485-5p_R + 1/BMP7 axis. Moreover, in vivo experiments in rats demonstrated that the overexpression of circ-SPATA13 in PDLSCs was associated with the promotion of bone formation in a skull defect model system.

Conclusion

These data supported the osteogenic functions of circ-SPATA13 in PDLSCs. Mechanistically, this circRNA was found to function as a molecular sponge for miR-485-5p_R + 1, in turn targeting BMP7 to promote the osteogenic differentiation of PDLSCs. This circ-SPATA13/miR-485-5p_R + 1/BMP7 axis may be a novel target for treatments promoting PDLSCs osteogenic differentiation.