Sun Xuefei, Meng Xiujiao, Piao Yu, Dong Shaojie, Dong Qianqian
Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, China.
Clinical Research Center of Shaanxi Province for Dental and Maxillofacial Diseases, Department of Endodontics, College of Stomatology, Xi'an Jiaotong University, Xi'an, China.
J Periodontal Res. 2024 Oct;59(5):1017-1030. doi: 10.1111/jre.13297. Epub 2024 Jun 4.
N6-Methyladenosine (mA) has been confirmed to play a dynamic role in osteoporosis and bone metabolism. However, whether mA is involved in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) remains unclear. The present study aimed to verify the role of methyltransferase-like 3 (METTL3)-mediated mA modification in the osteogenic differentiation of hPDLCs.
The METTL3, Runx2, Osx, and YAP mRNA expression was determined by qPCR. METTL3, RUNX2, OSX, YTHDF1, YAP, IGF2BP1, and eIF3a protein expression was measured by Western blotting and immunofluorescence assays. The levels of mA modification were evaluated by methylated RNA immunoprecipitation (MeRIP) and dot blot analyses. MeRIP-seq and RNA-seq were used to screen potential candidate genes. Nucleic acid and protein interactions were detected by immunoprecipitation. Alizarin red staining was used to evaluate the osteogenic differentiation of hPDLCs. Gene transcription and promoter activities were assessed by luciferase reporter assays (n ≥ 3).
The expression of METTL3 and mA modifications increased synchronously with the osteogenic differentiation of hPDLCs (p = .0016). YAP was a candidate gene identified by MeRIP-seq and RNA-seq, and its mRNA and protein expression levels were simultaneously increased. METTL3 increased the mA methylated IGF2BP1-mediated stability of YAP mRNA (p = .0037), which in turn promoted osteogenic differentiation (p = .0147). Furthermore, METTL3 increased the translation efficiency of YAP by recruiting YTHDF1 and eIF3a to the translation initiation complex (p = .0154), thereby promoting the osteogenic differentiation of hPDLCs (p = .0012).
Our study revealed that METTL3-initiated mA mRNA methylation promotes osteogenic differentiation of hPDLCs by increasing IGF2BP1-mediated YAP mRNA stability and recruiting YTHDF1 and eIF3a to the translation initiation complex to increase YAP mRNA translation. Our findings reveal the mechanism of METTL3-mediated mA modification during hPDLC osteogenesis, providing a potential therapeutic target for periodontitis and alveolar bone defects.
N6-甲基腺苷（mA）已被证实对骨质疏松症和骨代谢具有动态作用。然而，mA是否参与人牙周膜细胞（hPDLCs）的成骨分化仍不清楚。本研究旨在验证甲基转移酶样3（METTL3）介导的mA修饰在hPDLCs成骨分化中的作用。
通过qPCR测定METTL3、Runx2、Osx和YAP的mRNA表达。通过蛋白质免疫印迹法和免疫荧光分析测定METTL3、RUNX2、OSX、YTHDF1、YAP、IGF2BP1和eIF3a的蛋白表达。通过甲基化RNA免疫沉淀（MeRIP）和斑点印迹分析评估mA修饰水平。使用MeRIP-seq和RNA-seq筛选潜在的候选基因。通过免疫沉淀检测核酸和蛋白质相互作用。采用茜素红染色评估hPDLCs的成骨分化。通过荧光素酶报告基因测定评估基因转录和启动子活性（n≥3）。
METTL3的表达和mA修饰随着hPDLCs的成骨分化同步增加（p = 0.0016）。YAP是通过MeRIP-seq和RNA-seq鉴定的候选基因，其mRNA和蛋白表达水平同时增加。METTL3增加了mA甲基化的IGF2BP1介导的YAP mRNA稳定性（p = 0.0037），进而促进成骨分化（p = 0.0147）。此外，METTL3通过将YTHDF1和eIF3a募集到翻译起始复合物来提高YAP的翻译效率（p = 0.0154），从而促进hPDLCs的成骨分化（p = 0.0012）。
我们的研究表明，METTL3启动的mA mRNA甲基化通过增加IGF2BP1介导的YAP mRNA稳定性以及将YTHDF1和eIF3a募集到翻译起始复合物以增加YAP mRNA翻译来促进hPDLCs的成骨分化。我们的研究结果揭示了METTL3介导的mA修饰在hPDLCs成骨过程中的机制，为牙周炎和牙槽骨缺损提供了潜在的治疗靶点。
