METTL14 Regulates Autophagy and Osteogenic Differentiation of BMSCs Targeting Beclin-1 via an M ⁶ A-IGF2BPs-Dependent Mechanism

Background: The development of osteoporosis is often accompanied by autophagy disturbance, which also caused new osteoblasts defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. We aimed to explore the biological functions of autophagy mediated by METTL14 in osteoporosis and the possibility of METTL14 as an effective target for the treatment of osteoporosis. Methods: Calcein staining and microcomputed tomography (μ-CT) were used to analyze the bone formation. immunohistochemical (IHC) staining was used to detect the expression of METTL14 and beclin-1 in bone tissues. The quantitative real time PCR (qRT-PCR) and western blotting were used to analyze the mRNA and protein expressions. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were used to explore the osteogenic differentiation of BMSCs. The production of autophagy was determined by transmission electron microscopy (TEM) and mRFP-GFP-LC3 adenoviral. Moreover, RNA binding protein immunoprecipitation (RIP)-qPCR was used to investigate the m6A modification of beclin-1 and the binding capability of beclin-1 and IGF2BPs. Findings: Herein, we demonstrate that the expression of METTL14 is downregulated during the development of osteoporosis both in human and mice. In vivo, METTL14+/- knockdown mice exhibits elevated bone loss and impaired autophagy similar to the ovariectomized (OVX) mice, while injecting METTL14 overexpressing adenovirus delays the process of osteoporosis.In vitro, forced expression of METTL14 significantly facilitates the osteogenic differentiation ability of BMSCs through regulating the activation of autophagy by enhancing the expression of beclin-1 dependent on the m6 A modification; the opposite is true under METTL14 silencing condition. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A methylated beclin-1 mRNA and promotes its translation via mediating RNA stabilization. Interpretation: Our study reveals the METTL14-IGF2BPs-beclin-1 signaling axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14 mediated m 6 A modification in osteoporosis. Funding: This work was supported by grants from the National Natural Science Fund of China (81972117), Natural Science Foundation of Heilongjiang Province of China for excellent youth (JJ2020JQ0004), The First Affiliated Hospital of Harbin Medical University Excellent Young Talents Funding (HYD2020JQ0013), Special fund for clinical research of Wu Jieping Medical Foundation (320.6750.2020-04-50), and CAMS Innovation Fund for Medical Sciences (CIFMS, 2020-I2M-5-003). Declaration of Interest: The authors declare that they have no competing interests. Date Availability Ethical Approval: The experiments were performed under the guidance of the Experimental Animal Ethic Committee of the Harbin Medical University.