Bioinformatics identification and experimental validation of m6A-related diagnostic biomarkers in the subtype classification of blood monocytes from postmenopausal osteoporosis patients

Front Endocrinol (Lausanne). 2023 Mar 8:14:990078. doi: 10.3389/fendo.2023.990078. eCollection 2023.

Abstract

Background: Postmenopausal osteoporosis (PMOP) is a common bone disorder. Existing study has confirmed the role of exosome in regulating RNA N6-methyladenosine (m6A) methylation as therapies in osteoporosis. However, it still stays unclear on the roles of m6A modulators derived from serum exosome in PMOP. A comprehensive evaluation on the roles of m6A modulators in the diagnostic biomarkers and subtype identification of PMOP on the basis of GSE56815 and GSE2208 datasets was carried out to investigate the molecular mechanisms of m6A modulators in PMOP.

Methods: We carried out a series of bioinformatics analyses including difference analysis to identify significant m6A modulators, m6A model construction of random forest, support vector machine and nomogram, m6A subtype consensus clustering, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between different m6A patterns, principal component analysis, and single sample gene set enrichment analysis (ssGSEA) for evaluation of immune cell infiltration, experimental validation of significant m6A modulators by real-time quantitative polymerase chain reaction (RT-qPCR), etc.

Results: In the current study, we authenticated 7 significant m6A modulators via difference analysis between normal and PMOP patients from GSE56815 and GSE2208 datasets. In order to predict the risk of PMOP, we adopted random forest model to identify 7 diagnostic m6A modulators, including FTO, FMR1, YTHDC2, HNRNPC, RBM15, RBM15B and WTAP. Then we selected the 7 diagnostic m6A modulators to construct a nomogram model, which could provide benefit with patients according to our subsequent decision curve analysis. We classified PMOP patients into 2 m6A subtypes (clusterA and clusterB) on the basis of the significant m6A modulators via a consensus clustering approach. In addition, principal component analysis was utilized to evaluate the m6A score of each sample for quantification of the m6A subgroups. The m6A scores of patients in clusterB were higher than those of patients in clusterA. Moreover, we observed that the patients in clusterA had close correlation with immature B cell and gamma delta T cell immunity while clusterB was linked to monocyte, neutrophil, CD56dim natural killer cell, and regulatory T cell immunity, which has close connection with osteoclast differentiation. Notably, m6A modulators detected by RT-qPCR showed generally consistent expression levels with the bioinformatics results.

Conclusion: In general, m6A modulators exert integral function in the pathological process of PMOP. Our study of m6A patterns may provide diagnostic biomarkers and immunotherapeutic strategies for future PMOP treatment.

Keywords: RNA N6-methyladenosine (m6A) modulators; experimental validation; postmenopausal osteoporosis; risk prediction; subtype classification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO
  • Biomarkers
  • Computational Biology
  • Female
  • Fragile X Mental Retardation Protein
  • Humans
  • Monocytes
  • Osteoporosis*
  • Osteoporosis, Postmenopausal* / diagnosis
  • Osteoporosis, Postmenopausal* / genetics

Substances

  • Biomarkers
  • FMR1 protein, human
  • Fragile X Mental Retardation Protein
  • FTO protein, human
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO

Grants and funding

The project was generously supported by the grants from National Natural Science Foundation of China (82274542, 82274615, 81904225, 82205137 and 82205230), Guangdong Natural Science Foundation (2022A1515012062 and 2021A1515011247), Innovative Team Project and Key Project of the Department of Education of Guangdong Province (2021KCXTD017), High-Level University Collaborative Innovation Team of Guangzhou University of Chinese Medicine (2021xk57), Medical Research Foundation of Guangdong Province (A2021320), Guangzhou Science and Technology Project (202201020307), Scientific Research Project of Excellent Young Scholars Project of First Affiliated Hospital of Guangzhou University of Chinese Medicine (2019QN17), Scientific Research Project of Traditional Chinese Medicine Bureau of Guangdong Province (20201097 and 20221308). The funding institutions had not any role in the study design, data collection, data analysis, interpretation, or writing of the report in this study.