Identification and experimental validation of key m6A modification regulators as potential biomarkers of osteoporosis.

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Osteoporosis (OP) is a severe systemic bone metabolic disease that occurs worldwide. During the coronavirus pandemic, prioritization of urgent services and delay of elective care attenuated routine screening and monitoring of OP patients. There is an urgent need for novel and effective screening diagnostic biomarkers that require minimal technical and time investments. Several studies have indicated that N6-methyladenosine (m6A) regulators play essential roles in metabolic diseases, including OP. The aim of this study was to identify key m6A regulators as biomarkers of OP through gene expression data analysis and experimental verification. GSE56815 dataset was served as the training dataset for 40 women with high bone mineral density (BMD) and 40 women with low BMD. The expression levels of 14 major m6A regulators were analyzed to screen for differentially expressed m6A regulators in the two groups. The impact of m6A modification on bone metabolism microenvironment characteristics was explored, including osteoblast-related and osteoclast-related gene sets. Most m6A regulators and bone metabolism-related gene sets were dysregulated in the low-BMD samples, and their relationship was also tightly linked. In addition, consensus cluster analysis was performed, and two distinct m6A modification patterns were identified in the low-BMD samples. Subsequently, by univariate and multivariate logistic regression analyses, we identified four key m6A regulators, namely, METTL16, CBLL1, FTO, and YTHDF2. We built a diagnostic model based on the four m6A regulators. CBLL1 and YTHDF2 were protective factors, whereas METTL16 and FTO were risk factors, and the ROC curve and test dataset validated that this model had moderate accuracy in distinguishing high- and low-BMD samples. Furthermore, a regulatory network was constructed of the four hub m6A regulators and 26 m6A target bone metabolism-related genes, which enhanced our understanding of the regulatory mechanisms of m6A modification in OP. Finally, the expression of the four key m6A regulators was validated in vivo and in vitro, which is consistent with the bioinformatic analysis results. Our findings identified four key m6A regulators that are essential for bone metabolism and have specific diagnostic value in OP. These modules could be used as biomarkers of OP in the future.