Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China.
Endodontic Department, School of Stomatology, Nanjing Medical University, Nanjing, China.
Int Endod J. 2024 Apr;57(4):431-450. doi: 10.1111/iej.14021. Epub 2024 Jan 19.
Human stem cells from the apical papilla (SCAPs) are an appealing stem cell source for tissue regeneration engineering. Circular RNAs (circRNAs) are known to exert pivotal regulatory functions in various cell differentiation processes, including osteogenesis of mesenchymal stem cells. However, few studies have shown the potential mechanism of circRNAs in the odonto/osteogenic differentiation of SCAPs. Herein, we identified a novel circRNA, circ-ZNF236 (hsa_circ_0000857) and found that it was remarkably upregulated during the SCAPs committed differentiation. Thus, in this study, we showed the significance of circ-ZNF236 in the odonto/osteogenic differentiation of SCAPs and its underlying regulatory mechanisms.
The circular structure of circ-ZNF236 was identified via Sanger sequencing, amplification of convergent and divergent primers. The proliferation of SCAPs was detected by CCK-8, flow cytometry analysis and EdU incorporation assay. Western blotting, qRT-PCR, Alkaline phosphatase (ALP) and Alizarin red staining (ARS) were performed to explore the regulatory effect of circ-ZNF236/miR-218-5p/LGR4 axis in the odonto/osteogenic differentiation of SCAPs in vitro. Fluorescence in situ hybridization, as well as dual-luciferase reporting assays, revealed that circ-ZNF236 binds to miR-218-5p. Transmission electron microscopy (TEM) and mRFP-GFP-LC3 lentivirus were performed to detect the activation of autophagy.
Circ-ZNF236 was identified as a highly stable circRNA with a covalent closed loop structure. Circ-ZNF236 had no detectable influence on cell proliferation but positively regulated SCAPs odonto/osteogenic differentiation. Furthermore, circ-ZNF236 was confirmed as a sponge of miR-218-5p in SCAPs, while miR-218-5p targets LGR4 mRNA at its 3'-UTR. Subsequent rescue experiments revealed that circ-ZNF236 regulates odonto/osteogenic differentiation by miR-218-5p/LGR4 in SCAPs. Importantly, circ-ZNF236 activated autophagy, and the activation of autophagy strengthened the committed differentiation capability of SCAPs. Subsequently, in vivo experiments showed that SCAPs overexpressing circ-ZNF236 promoted bone formation in a rat skull defect model.
Circ-ZNF236 could activate autophagy through increasing LGR4 expression, thus positively regulating SCAPs odonto/osteogenic differentiation. Our findings suggested that circ-ZNF236 might represent a novel therapeutic target to prompt the odonto/osteogenic differentiation of SCAPs.
人根尖乳头干细胞（SCAPs）是组织再生工程中一种有吸引力的干细胞来源。环状 RNA（circRNAs）已知在多种细胞分化过程中发挥关键的调节作用，包括间充质干细胞的成骨作用。然而，很少有研究表明 circRNAs 在 SCAPs 的牙/骨向分化中的潜在机制。在此，我们鉴定了一种新型 circRNA，circ-ZNF236（hsa_circ_0000857），并发现它在 SCAPs 定向分化过程中显著上调。因此，在本研究中，我们展示了 circ-ZNF236 在 SCAPs 牙/骨向分化中的重要性及其潜在的调控机制。
通过 Sanger 测序、趋同和发散引物扩增鉴定 circ-ZNF236 的环状结构。通过 CCK-8、流式细胞术分析和 EdU 掺入实验检测 SCAPs 的增殖。Western blot、qRT-PCR、碱性磷酸酶（ALP）和茜素红染色（ARS）实验用于研究 circ-ZNF236/miR-218-5p/LGR4 轴在 SCAPs 体外牙/骨向分化中的调控作用。荧光原位杂交以及双荧光素酶报告实验表明 circ-ZNF236 与 miR-218-5p 结合。透射电子显微镜（TEM）和 mRFP-GFP-LC3 慢病毒用于检测自噬的激活。
鉴定出 circ-ZNF236 是一种具有共价闭合环结构的高度稳定的 circRNA。circ-ZNF236 对细胞增殖没有明显影响，但能正向调控 SCAPs 的牙/骨向分化。此外，circ-ZNF236 在 SCAPs 中被证实为 miR-218-5p 的海绵，而 miR-218-5p 在其 3'UTR 上靶向 LGR4 mRNA。随后的挽救实验表明，circ-ZNF236 通过 miR-218-5p/LGR4 在 SCAPs 中调节牙/骨向分化。重要的是，circ-ZNF236 通过增加 LGR4 的表达激活自噬，从而正向调节 SCAPs 的牙/骨向分化。随后，体内实验表明，过表达 circ-ZNF236 的 SCAPs 在大鼠颅骨缺损模型中促进骨形成。
circ-ZNF236 通过增加 LGR4 的表达激活自噬，从而正向调节 SCAPs 的牙/骨向分化。我们的研究结果表明，circ-ZNF236 可能成为一种新的治疗靶点，以促进 SCAPs 的牙/骨向分化。
