Methyltransferase-like 3 (METTL3) is the main enzyme for N⁶-methyladenosine (m⁶A)-based methylation of RNAs and it has been implicated in many biological and pathophysiological processes. In this study, we aimed to explore the potential involvement of METTL3 in osteoblast differentiation and decipher the underlying cellular and molecular mechanisms. We demonstrated that METTL3 is downregulated in human osteoporosis and the ovariectomized (OVX) mouse model, as well as during the osteogenic differentiation. Silence of METTL3 by short interfering RNA (siRNA) decreased m⁶A methylation levels and inhibited osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and reduced bone mass, and similar effects were observed in METTL3^+/− knockout mice. In contrast, adenovirus-mediated overexpression of METTL3 produced the opposite effects. In addition, METTL3 enhanced, whereas METTL3 silence or knockout suppressed, the m⁶A methylations of runt-related transcription factor 2 (RUNX2; a key transcription factor for osteoblast differentiation and bone formation) and precursor (pre-)miR-320. Moreover, downregulation of mature miR-320 rescued the decreased bone mass caused by METTL3 silence or METTL3^+/− knockout. Therefore, METTL3-based m⁶A modification favors osteogenic differentiation of BMSCs through m⁶A-based direct and indirect regulation of RUNX2, and abnormal downregulation of METTL3 is likely one of the mechanisms underlying osteoporosis in patients and mice. Thus, METTL3 overexpression might be considered a new approach of replacement therapy for the treatment of human osteoporosis.

甲基转移酶样3（METTL3）是基于N ^6-甲基腺苷（m ⁶ A）的RNA甲基化的主要酶，它已涉及许多生物学和病理生理过程。在这项研究中，我们旨在探讨METTL3在成骨细胞分化中的潜在参与并破译潜在的细胞和分子机制。我们证明了METTL3在人类骨质疏松症和卵巢切除（OVX）小鼠模型中以及在成骨分化过程中均下调。短干扰RNA（siRNA）使METTL3沉默，降低了m ⁶ A甲基化水平，抑制了骨髓间充质干细胞（BMSCs）的成骨分化并降低了骨量，在METTL3中也观察到了类似的作用^+/-敲除小鼠。相反，腺病毒介导的METTL3的过表达产生相反的作用。此外，METTL3增强，而METTL3沉默或敲除则受到抑制，这是矮子相关转录因子2（RUNX2；成骨细胞分化和骨形成的关键转录因子）和前体（pre）miR-320的m ⁶ A甲基化。此外，成熟miR-320的下调可以挽救由METTL3沉默或METTL3 ^+/-敲除引起的骨量减少。因此，基于METTL3的m ⁶ A修饰有助于通过m ⁶促进BMSC的成骨分化基于A的RUNX2的直接和间接调节以及METTL3的异常下调可能是患者和小鼠骨质疏松症的潜在机制之一。因此，METTL3过表达可能被认为是替代疗法治疗人骨质疏松症的一种新方法。