Sui Meizhi, Lyu Jiaxuan, Zhou Jiaxin, Liao Qian, Xiao Zexu, Jin Mingming, Tao Jiang
Department of General Dentistry, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200011, China.
Department of Stomatology, Kashi Prefecture Second People's Hospital, No 1, Jiankang Road, Kashi, 844000, Xinjiang, China.
Stem Cell Res Ther. 2025 Mar 13;16(1):137. doi: 10.1186/s13287-025-04239-z.
Circular RNAs (circRNAs) play a crucial role in stem cell-based tooth regeneration. However, the functions and underlying mechanisms of circRNAs in tooth regeneration from human dental pulp stem cells (DPSCs) remain largely unclear.
In this study, DPSCs were used for odontogenic differentiation. High-throughput sequencing was performed for differential circRNA analysis. A luciferase reporter assay was conducted to confirm the downstream target of the circRNA, circ-AARS. We then constructed vectors and siRNAs for overexpressing and silencing circ-AARS, miR-24-3p, and Krüppel-like factor 6 (KLF6) and transfected them into DPSCs. Alkaline phosphatase staining, Alizarin Red S staining, western blotting assay, and quantitative reverse transcription-polymerase chain reaction were used to explore the underlying mechanisms of circ-AARS. Finally, a heterotopic bone model was utilized to reveal the regulating effects of circ-AARS.
High-throughput sequencing analysis showed that circ-AARS plays an important role during the odontogenic differentiation of DPSCs. Downregulation of circ-AARS inhibited the odontogenic differentiation of DPSCs; however, circ-AARS overexpression promoted their odontogenic differentiation. Bioinformatics analysis and luciferase reporter assay confirmed that both miR-24-3p and KLF6 were the downstream targets of circ-AARS. miR-24-3p downregulation or KLF6 overexpression restored the odontogenic differentiation ability of DPSCs after circ-AARS silencing. KLF6 upregulation restored the odontogenic differentiation ability of DPSCs after KLF6 overexpression. The heterotopic bone model confirmed that circ-AARS overexpression promoted the odontoblastic differentiation of DPSCs.
The present study showed that circ-AARS can promote the odontoblastic differentiation of DPSCs by increasing KLF6 expression and sponging miR-24-3p. Taken together, the results indicate that circ-AARS may be a potential positive regulator of odontoblastic differentiation of DPSCs.
环状RNA（circRNAs）在基于干细胞的牙齿再生中起着关键作用。然而，circRNAs在人牙髓干细胞（DPSCs）牙齿再生中的功能及潜在机制仍不清楚。
在本研究中，使用DPSCs进行成牙分化。进行高通量测序以分析差异circRNA。进行荧光素酶报告基因检测以确认circRNA circ-AARS的下游靶点。然后构建用于过表达和沉默circ-AARS、miR-24-3p和Krüppel样因子6（KLF6）的载体和小干扰RNA（siRNAs），并将它们转染到DPSCs中。采用碱性磷酸酶染色、茜素红S染色、蛋白质印迹分析和定量逆转录-聚合酶链反应来探究circ-AARS的潜在机制。最后，利用异位骨模型揭示circ-AARS的调节作用。
高通量测序分析表明circ-AARS在DPSCs成牙分化过程中起重要作用。circ-AARS的下调抑制了DPSCs的成牙分化；然而circ-AARS的过表达促进了它们的成牙分化。生物信息学分析和荧光素酶报告基因检测证实miR-24-3p和KLF6均为circ-AARS的下游靶点。circ-AARS沉默后，miR-24-3p的下调或KLF6的过表达恢复了DPSCs的成牙分化能力。KLF6过表达后，KLF6的上调恢复了DPSCs的成牙分化能力。异位骨模型证实circ-AARS的过表达促进了DPSCs的成牙本质细胞分化。
本研究表明circ-AARS可通过增加KLF6表达和吸附miR-24-3p来促进DPSCs的成牙本质细胞分化。综上所述，结果表明circ-AARS可能是DPSCs成牙本质细胞分化的潜在正调节因子。
