m6A Methylation of Precursor-miR-320/RUNX2 Controls Osteogenic Potential of Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

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.

Multifaceted Functions and Novel Insight Into the Regulatory Role of RNA N6-Methyladenosine Modification in Musculoskeletal Disorders

“…In the first place, knockdown of m 6 A methyltransferase METTL3 in mice induced pathological features related to the occurrence of OP via decreasing parathyroid hormone (PTH)/parathyroid hormone receptor-1 (Pth1r) signaling axis, interfering the PTH-induced osteogenic response of BMSCs ( Wu et al, 2018 ). Moreover, downregulation of METTL3 in BMSCs inhibited the methylation of runt-related transcription factor 2 (RUNX2) and precursor (pre-) miR320 ( Yan et al, 2020 ). RUNX2 is an essential regulator of osteoblast progenitor proliferation and osteogenic differentiation, which can enhance the bone mineralization, and multiple factors were reported to participate in the osteogenic process via targeting RUNX2 ( Hou et al, 2019 ; Komori, 2019 ).…”

MiR‐103‐3p targets the m6A methyltransferase METTL14 to inhibit osteoblastic bone formation

“…Previous studies have suggested that altered METTL3/m 6 A modification could participate in regulation of miRNAs in many biological processes (Alarcón et al, 2015; Han et al, 2019; Peng et al, 2019; Wang, Deng, et al, 2019; Wang, Ishfaq, et al, 2019; Yan et al, 2020; Zhang, Bai, et al, 2019). To identify whether altered METTL14/m 6 A modification is involved in regulating miR‐103‐3p in an m 6 A‐dependent pri‐miRNA‐processing manner, we first examined whether METTL14 was required for engagement of pri‐miRNAs by the microprocessor protein DGCR8.…”

Biological functions of m6A methyltransferases

“…Thirdly, the bio-function of m 6 A methyltransferase remains contradictory. For instance, in osteogenesis, while most scientists have proved that METTL3 expression in BMScs is positively correlated with osteogenesis and negatively correlated with adipogenesis [51,52], Yu J et al revealed that METTL3 inhibits the calcium deposition and alkaline phosphatase activity of BMSCs and attenuates the activation of NF-κB to impair osteogenesis [55]. In another example, METTL3 exhibited an inhibitory role in the proliferation, tumorigenicity and migration ability in glioma cells [144], which had a paradoxical effect [149].…”

“…On the one hand, METTL3 expression positively correlates with BMSC-driven osteogenesis. Conditional deletion of METTL3 in BMSCs resulted in incompetent osteogenic differentiation potential, reduced bone mass and impaired bone formation [ 51 , 52 ] (Fig. 1 c).…”

“…METTL3 knockdown decreased the expression of bone formation-related genes (such as Runx2 and Osterix), precursor (pre-) miR-320 and the PI3K-Akt pathway. Furthermore, the activity of alkaline phosphatase (ALP), the formation of mineralized nodules, the expression of Vegfa and its splice variants vegfa-164 and vegfa-188 were also influenced [ 52 , 53 ] (Fig. 1 c).…”