M6A Methyltransferase WTAP Regulates BMSC Differentiation And Its Mechanism In Osteoporosis

BackgroundOsteoporosis(OP)is a systemic bone metabolic disease characterized by the destruction of the microstructure of bone tissue and the reduction of bone mass,which leads to increased brittleness of bone and increase the risk of fracture.OP and fractures have become a major cause of disability and death among the elderly,which will become a serious social problem as the world population ages.Epidemiological studies show that there are more than 900 million osteoporotic fractures worldwide.Nearly 200 million people in the United States suffer osteoporotic fractures each year,and the direct cost of osteoporotic fractures is more than $17 billion annually.It is predicted that the burden will increase by 50%by 2025.Therefore,osteoporosis and osteoporotic fractures caused by aging are urgent problem.Clarifying the pathogenesis of osteoporosis and seeking ideal diagnosis and treatment targets is of great significance for improving the quality of life of osteoporosis patients.In recent years,epigenetic modification has been widely studied in various fields.As the most abundant RNA modification mode in eukaryotes,m6A(N6-methyladenosine)methylation has become a new research hotspot.Studies have found that m6A modification presents dynamic and reversible changes in different developmental stages and tissues.Moreover,methylation transferase(METTL3,METTL14,WTAP,KIAA1429),methylation recognition enzyme(HNRNPC,YTHDF1,YTHDF2,YTHDF3,YTHDC1)and demethylation enzyme(FTO,ALKBH5)are required to participate together.RNA m6A modification is related to cell growth and development,tumor migration ability,regulation of metabolic diseases,cardiovascular repair and circadian rhythm regulation.However,as a key component of the m6A methylated transferase complex,the mechanism of Wilms tumor 1-associated protein(WTAP)in OP has not been studied.Based on the above scientific questions,we designed and conducted this study to explore the role,function and specific mechanism of WTAP-mediated m6A RNA methylation modification in OP.Objective1.To explore the expression of m6A methyltransferase WTAP in osteoporotic bone tissue and the differentiation of BMSCs.2.To elucidate the role of WTAP-mediated m6A RNA methylation modification in osteogenic differentiation and lipogenic differentiation of BMSCs.3.To explore the mechanism of WTAP in the differentiation of BMSCs,to add a new research basis for clarifying the pathogenesis and therapeutic targets of osteoporosis,and to provide new ideas for clinical exploration of effective intervention strategies for the treatment of OP progress.Methods1.Western Blot,RT-qPCR and m6A colorimetric assay were used to detect the expression of WTAP and m6A in bone tissue of osteoporosis patients,bone tissue of osteoporosis mouse model and corresponding normal bone tissue.2.Western Blot,RT-qPCR and m6A colorimetric assay were used to verify the expressions of WTAP and m6A during osteogenic and adipogenic differentiation of BMSCs.3.Western Blot,RT-qPCR,alizarin red staining(ARS)and alkaline phosphatase staining(ALP staining)were used to detect the effect of WTAP overexpression or WTAP knockdown on the BMSCs differentiation.4.Micro-CT,HE and Masson staining were used to detect the effect of WTAP on bone mass in mice with osteoporosis.5.micro-RNA sequencing was used to analyze the differentially expressed genes between groups,and to detect the effect of WTAP overexpression on downstream genes during BMSCs differentiation.6.The interaction between the downstream target genes microRNA and WTAP was verified by RNA binding protein immunoprecipitation and m6A immunoprecipitation.7.Western Blot,RT-qPCR,ARS staining and ALP staining were used to verify the direct effect of microRNA on BMSCs differentiation.8.Bioinformatics predicted the downstream target genes of microRNA,and the interaction between microRNA and target genes was verified by biluciferase experiment.Results1.Western Blot,RT-qPCR and m6A colorimetric assay showed that compared with normal bone tissue,the expression levels of WTAP and m6A in bone tissue of osteoporosis were significantly decreased.2.In vitro experiments showed that the expressions of m6A and WTAP were highly expressed during osteogenic differentiation and low expressed during adipogenic differentiation of BMSCs.3.Overexpression of WTAP can promote osteogenic differentiation of BMSC and inhibit adipogenic differentiation of BMSCs.WTAP Knockdown showed the opposite result.4.In vivo results showed that upregulation of WTAP reduced bone mass loss in mice with osteoporosis.5.micro-RNA sequencing analysis showed that the downstream target gene of WTAP was miR-29b-3p.6.The results of RNA-binding protein immunoprecipitation and m6A immunoprecipitation showed that WTAP-mediated m6A could act on the downstream target gene miR-29b-3p.7.Overexpression of miR-29b-3p can promote osteogenic differentiation of BMSCs and inhibit adipogenic differentiation of BMSCs.8.Dual luciferase assay showed that miR-29b-3p regulates BMSCs differentiation by targeting HDAC4.Conclusion1.The expression levels of WTAP and m6A are significantly down-regulated in bone tissue of osteoporosis,and the changes of WTAP and m6A can affect the osteogenic differentiation and adipogenic differentiation of BMSCs.2.WTAP-mediated m6A methylation affects the osteogenic differentiation and adipogenic differentiation of BMSCs by regulating the maturation of miR-29b-3p.3.WTAP-mediated m6A methylation regulates the osteogenic differentiation and adipogenic differentiation of BMSCs through the miR-29b-3p/HDAC4 signaling axis,ultimately affecting the occurrence and development of osteoporosis.