环状 RNA 相互作用蛋白激酶 3 通过 miR-10b-5p/DOHH/NF-κB 轴调控成牙骨质细胞分化。
CircHIPK3 regulates cementoblast differentiation via the miR-10b-5p/DOHH/NF-κB axis.
机构信息
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, China.
State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, China; Department of Periodontology, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
出版信息
Cell Signal. 2024 Dec;124:111427. doi: 10.1016/j.cellsig.2024.111427. Epub 2024 Sep 18.
BACKGROUND
Intact cementum is vital for tooth stability and health. Cementoblasts, which line the root surface, are responsible for cementum formation. Recent evidence suggests that circular RNAs (circRNAs) are involved in various cellular functions and may have clinical applications. Although circHIPK3 has been shown to participate in osteogenesis, its role in cementoblast differentiation and mineralization is not well understood.
METHODS
The ring structure of circHIPK3 was confirmed using Sanger sequencing and an actinomycin D assay. Subcellular localization of circHIPK3 was assessed using a nucleus-cytoplasm separation assay. RT-qPCR was employed to analyze circHIPK3 expression during cementoblast differentiation and following TNF-α treatment. In vivo, periapical lesions were induced in mouse mandibular first molars to mimic an inflammatory environment, and circHIPK3 expression was evaluated. The interaction of the circHIPK3/miR-10b-5p/DOHH axis was explored through RNA pull-down assays, bioinformatics analysis, and dual-luciferase reporter assays. The effects on cementoblast differentiation and mineralization were assessed by measuring osteogenic markers, alkaline phosphatase (ALP) activity, ALP staining, and alizarin red S staining.
RESULTS
CircHIPK3 was predominantly located in the cytoplasm of cementoblasts, and its expression was significantly upregulated during cementoblast differentiation. Knockdown of circHIPK3 inhibited cementoblast differentiation and mineralization, whereas its overexpression promoted these processes. Mechanistically, circHIPK3 upregulated DOHH expression by sponging miR-10b-5p, thereby enhancing cementoblast differentiation and mineralization. The NF-κB pathway was found to act downstream of the circHIPK3/miR-10b-5p/DOHH axis in these processes. Additionally, circHIPK3 expression was significantly downregulated in inflammatory environments both in vitro and in vivo. Forced overexpression of circHIPK3 mitigated the inhibitory effects of TNF-α on cementoblast differentiation and mineralization.
CONCLUSION
CircHIPK3 acts as a positive regulator of cementoblast differentiation and mineralization through the miR-10b-5p/DOHH/NF-κB axis, playing a crucial role in both normal and pathological cementogenesis.
背景
完整的牙骨质对于牙齿的稳定性和健康至关重要。成牙骨质细胞排列在牙根表面，负责牙骨质的形成。最近的证据表明，环状 RNA（circRNA）参与各种细胞功能，并且可能具有临床应用价值。虽然 circHIPK3 已被证明参与成骨作用，但它在成牙骨质细胞分化和矿化中的作用尚不清楚。
方法
通过 Sanger 测序和放线菌素 D 测定证实了 circHIPK3 的环状结构。通过核质分离测定评估 circHIPK3 的亚细胞定位。使用 RT-qPCR 分析成牙骨质细胞分化过程中和 TNF-α处理后 circHIPK3 的表达。在体内，通过诱导小鼠下颌第一磨牙根尖周病变来模拟炎症环境，并评估 circHIPK3 的表达。通过 RNA 下拉测定、生物信息学分析和双荧光素酶报告基因测定探讨了 circHIPK3/miR-10b-5p/DOHH 轴的相互作用。通过测量成骨标志物、碱性磷酸酶（ALP）活性、ALP 染色和茜素红 S 染色来评估对成牙骨质细胞分化和矿化的影响。
结果
circHIPK3 主要位于成牙骨质细胞的细胞质中，其表达在成牙骨质细胞分化过程中显著上调。circHIPK3 敲低抑制成牙骨质细胞分化和矿化，而过表达则促进这些过程。机制上，circHIPK3 通过海绵吸附 miR-10b-5p 而上调 DOHH 的表达，从而增强成牙骨质细胞的分化和矿化。在这些过程中，NF-κB 途径被发现位于 circHIPK3/miR-10b-5p/DOHH 轴的下游。此外，circHIPK3 的表达在体外和体内炎症环境中均显著下调。强制过表达 circHIPK3 减轻了 TNF-α对成牙骨质细胞分化和矿化的抑制作用。
结论
circHIPK3 通过 miR-10b-5p/DOHH/NF-κB 轴作为成牙骨质细胞分化和矿化的正调节剂发挥作用，在正常和病理性牙骨质形成中都起着至关重要的作用。
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