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免疫沉淀
信使核糖核酸
化学
细胞生物学
污渍
分子生物学
免疫印迹
核糖核酸
基因表达
牙周膜干细胞
碱性磷酸酶
生物
基因
生物化学
酶
作者
Xuefei Sun,Xiujiao Meng,Y. Piao,Shaojie Dong,Qianqian Dong
摘要
Abstract Aims N6‐Methyladenosine (m 6 A) has been confirmed to play a dynamic role in osteoporosis and bone metabolism. However, whether m 6 A 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 m 6 A modification in the osteogenic differentiation of hPDLCs. Methods 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 m 6 A 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). Results The expression of METTL3 and m 6 A 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 m 6 A 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). Conclusion Our study revealed that METTL3‐initiated m 6 A 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 m 6 A modification during hPDLC osteogenesis, providing a potential therapeutic target for periodontitis and alveolar bone defects.
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