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.

The development of osteoporosis is often accompanied by autophagy disturbance, which also causes new osteoblast 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. Herein, we demonstrate that METTL14 plays a critical role in autophagy induction and hinders osteoporosis process whose expression is decreased both in human osteoporosis bone tissue and ovariectomy (OVX) mice. In vivo, METTL14^+/− knockdown mice exhibit elevated bone loss and impaired autophagy similar to the OVX mice, while overexpression of METTL14 significantly promotes bone formation and inhibits the progression of osteoporosis caused by OVX surgery. In vitro, METTL14 overexpression significantly enhances the osteogenic differentiation ability of BMSCs through regulating the expression of beclin-1 depending on m⁶A modification and inducing autophagy; the opposite is true with METTL14 silencing. Subsequently, m⁶A-binding proteins IGF2BP1/2/3 recognize m⁶A-methylated beclin-1 mRNA and promote its translation via mediating RNA stabilization. Furthermore, METTL14 negatively regulates osteoclast differentiation. Collectively, our study reveals the METTL14/IGF2BPs/beclin-1 signal axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14-mediated m⁶A modification in osteoporosis.

Acute myocardial infarction (AMI) is the leading cause of sudden death worldwide. MicroRNA-155 (miR-155) has been reported to target antiapoptotic genes in various diseases models, but the functional role of miR-155 in response to MI injury needs further investigations. This study investigated the role of miR-155 in myocardial ischemia injury. TUNEL and flow cytometry were performed to measure cell apoptosis. Western blot analysis was employed to detect protein expressions of Bcl-2, XIAP, Bax, and caspase-3. qRT-PCR was used to quantify miRNA levels. We showed that miR-155 was dynamically elevated in murine hearts subjected to MI and in neonatal rat ventricular cardiomyocyte (NRVM) injury induced by hydrogen peroxide (H₂O₂). In response to H₂O₂, the silencing of miR-155 using AMO-155 (antisense inhibitor oligodeoxyribonucleotides) significantly increased cell viability and reduced cell apoptosis. Moreover, AMO-155 reversed the H₂O₂-induced downregulation of Bcl-2 and XIAP and upregulation of Bax and cleaved-caspase-3. Further study revealed that AMO-155 resulted in a decrease of H₂O₂-induced JC-1-labelled monomeric cell number. In addition, AMO-155 markedly decreased infarct size, ameliorated impaired cardiac function, and significantly reduced apoptotic cell percentages in MI mice heart. The RNA-binding protein Quaking (QKI) was predicted as a target gene of miR-155 through bioinformatic analysis, and AMO-155 attenuated the downregulation of QKI in H₂O₂-treated cardiomyocytes and MI mice heart. Knockdown of QKI by siRNA abolished the antiapoptotic effects of AMO-155. Taken together, miR-155 is upregulated in the MI heart and NRVMs in response to H₂O₂stress, and downregulating of miR-155 protects cardiomyocytes against apoptosis. Mechanistically, it is probably due to the repression of QKI signaling pathway.

The dynamic changes of RNA N6-methyladenosine (m⁶A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m⁶A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m⁶A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m⁶A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m⁶A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m⁶A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m⁶A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m⁶A-LDHA/PFKM in osteosarcoma.