Xiao Tong, Shi Yijia, Ye Yu, Wang Jing, Wang Wenmin, Yu Haowen, Yan Maoshen, Yu Jinhua
Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing 210029, China; State Key Laboratory Cultivation Base of Research, Prevention and Treatment for Oral Diseases, Nanjing Medical University, Nanjing 210029, China; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing Medical University, Nanjing 210029, China.
Department of Endodontics, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing 210029, China; State Key Laboratory Cultivation Base of Research, Prevention and Treatment for Oral Diseases, Nanjing Medical University, Nanjing 210029, China; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing Medical University, Nanjing 210029, China.
Cell Signal. 2025 Mar;127:111561. doi: 10.1016/j.cellsig.2024.111561. Epub 2024 Dec 10.
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.
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.
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.
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.
人牙周膜干细胞（PDLSCs）来源广泛且具有较强的成骨分化能力，这使其成为骨再生的理想工具。环状RNA（circRNAs）在细胞分化过程中发挥多种功能，是潜在的治疗靶点。在此，我们鉴定出一种新的circRNA，即circ-SPATA13，并发现它与PDLSCs的成骨分化呈高度正相关。因此，在本研究中，我们揭示了circ-SPATA13在PDLSCs成骨分化中的意义及机制。
从根尖发育不全的第三磨牙中分离出PDLSCs，并诱导其进行软骨、脂肪或成骨分化。通过流式细胞术检测表面标志物。用EdU和CCK-8法评估细胞增殖。通过茜素红和碱性磷酸酶染色评估circ-SPATA13和miR-485-5p_R + 1对矿物质沉积的调控作用。通过蛋白质免疫印迹法、免疫荧光法和qRT-PCR检测成骨相关因子的表达。采用荧光原位杂交法检测circ-SPATA13在PDLSCs内的定位。通过蛋白质免疫印迹法、qRT-PCR、双荧光素酶报告基因检测、拯救实验和生物信息学方法评估circ-SPATA13、miR-485-5p_R + 1和BMP7在PDLSCs成骨过程中的关系。
分离出表达间充质干细胞表面标志物的原代PDLSCs。鉴定出circ-SPATA13，发现其对PDLSCs的增殖无影响，而它是PDLSCs成骨分化的正向调节因子，这一过程受到miR-485-5p_R + 1的拮抗。双荧光素酶报告基因检测显示，circ-SPATA13能够作为分子海绵在PDLSCs内吸附miR-485-5p_R + 1，而该miRNA能够与靶mRNA BMP7的3'-UTR结合。在拯救实验中，证实circ-SPATA13通过该miR-485-5p_R + 1/BMP7轴调节PDLSCs的成骨分化。此外，大鼠体内实验表明，PDLSCs中circ-SPATA13的过表达与颅骨缺损模型系统中骨形成的促进有关。
这些数据支持circ-SPATA13在PDLSCs中的成骨功能。从机制上讲，发现该circRNA作为miR-485-5p_R + 1的分子海绵，进而靶向BMP7促进PDLSCs的成骨分化。该circ-SPATA13/miR-485-5p_R + 1/BMP7轴可能是促进PDLSCs成骨分化治疗的新靶点。
