MiR-103-3p Targets The M~6A Methyltransferase METTL14 To Inhibit Osteoblastic Bone Formation

BackgroundOsteoporosis is a common disease among the elderly and postmenopausal women.Emerging evidence indicates that osteoblasts directly affect the entire bone remodeling process and that impaired osteoblast activity plays fundamental roles in bone metabolic disorder.Moreover,the mechanisms that mediate impaired osteoblast activity remain unclear and merit further research.microRNAs(miRNAs)are small single-stranded noncoding RNA molecules involved in silencing and posttranscriptional regulation of gene expression,thereby mediating many biological processes.Many miRNAs have been characterized to regulate osteoblast activity and osteoblastic bone formation.Our previous study revealed that mature miR-103-3p was evolutionarily conserved among several species and highly expressed in bone tissue.In addition,we found that miR-103-3p could inhibit L-type calcium channel currents and osteoblast proliferation under simulated microgravity conditions,primarily by suppressing Cav1.2 expression.Other researchers showed that miR-103-3p was downregulated under cyclic mechanical loading conditions and found that this alteration was adversely correlated with osteoblast differentiation and bone formation in response to mechanical stimulation.However,the influence of miR-103-3p in osteoblastic bone formation in postmenopausal osteoporosis and the possible underlying mechanisms have not been confirmed.N~6-methyladenosine(m~6A)is the most prevalent epigenetic modification in eukaryotic mRNAs and regulates a broad spectrum of biological processes.In mammals,m~6A is installed by an m~6A methyltransferase complex consisting of methyltransferase-like 3(METTL3),methyltransferase-like 14(METTL14)and Wilms tumor 1-associated protein(WTAP)and can be removed by the demethylases alk B homologue 5(ALKBH5)and fat mass and obesity-associated protein(FTO).The m~6A methyltransferase METTL3 and demethylase FTO have been reported to regulate osteogenic activity and bone formation.However,the biological significance of the other m~6A modulators in bone formation or osteoporosis remains elusive.Methods1.Clinical samples were obtained and animal models were established.Real-time PCR was used to detect the levels of miR-103-3p and bone differentiation markers in clinical and model animal femur samples.And then we verify the correlation between miR-103-3p and bone differentiation markers.In addition,real-time PCR was used to detect the levels of miR-103-3p and bone differentiation markers during osteoblast differentiation.2.The mimics of miR-103-3p(agomir-103-3p)and the inhibitor of miR-103-3p,(antagmir-103-3p)were constructed.CCK-8 detection,EdU staining,real-time PCR,ELISA,ALP staining and Alizarin red staining were used to detect the effect of miR-103-3p on osteoblast osteogenic function in vitro.The effects of miR-103-3p on bone formation ability were measured by microCT,calcein staining,Masson's trichrome staining and three-point stress test in vivo.Luciferase reporter gene technology,Western blot and RIP analysis were used to verify the mechanism of miR-103-3p in regulating osteogenic activity.3.RIP analysis,m~6A content analysis and real-time PCR were used to verify the feedback regulation of METTL14 on miR-103-3p.CCK-8 assay,EdU staining,real-time PCR,ELISA,ALP staining and Alizarin red staining were used to detect the effect of METTL14 on osteoblast osteogenic function in vitro.4.Real-time PCR,Western blot and m~6A content analysis were used to detect the levels of METTL14,bone differentiation markers and m~6A content in clinical and animal model femur samples.And then we verify the correlation between miR-103-3p,METTL14,m~6A and bone differentiation markers.In addition,real-time PCR was used to detect the levels of METTL14,m~6A and bone differentiation markers during osteoblast differentiation.Results1.The miR-103-3p level increased in the femur of osteoporosis patients and ovariectomized mice,and the level of miR-103-3p was negatively correlated with the content of bone differentiation markers.In addition,the content of miR-103-3p decreased with the differentiation of primary osteoblasts.2.miR-103-3p targeted the m~6A methyltransferase METTL14 to inhibit osteoblastic bone formation.3.METTL14-dependent m~6A methylation regulated the processing of miR-103-3p and osteoblast activity in vitro.4.METTL14 levels and m~6A content in total RNA were lower in osteoporosis patients and ovariectomized mice.The contents of METTL14 and m~6A were negatively correlated with miR-103-3p level,and positively correlated with the content of bone differentiation markers.In addition,the contents of METTL14 and m~6A increased with the differentiation of primary osteoblasts.ConclusionsmiR-103-3p/METTL14/m~6A signal axis plays a key role in the process of osteoblast bone formation,which may become a potential therapeutic target for osteoporosis.