Multi-omics analysis identifies ANXA2-associated transcriptomic and splicing networks in osteoblast proliferation and apoptosis

Abstract Osteoporosis (OP), a prevalent metabolic bone disorder characterized by impaired osteoblast function, poses a major health burden worldwide. Annexin A2 (ANXA2), a multifunctional protein with RNA-binding activity, plays a role in OP progression by influencing osteoblast differentiation and apoptosis. However, as an RNA-binding protein (RBP), the genome-wide RNA targets and the molecular mechanisms by which ANXA2 governs osteoblast activity are largely unknown. In this study, we systematically investigated the downstream regulatory targets and associated proteins of ANXA2 in mouse osteoblasts (MC3T3-E1 cell line) using shRNA-mediated knockdown, RNA sequencing (RNA-seq), improved RNA-immunoprecipitation sequencing (iRIP-seq), and co-immunoprecipitation coupled with mass spectrometry (CO-IP/MS). Our findings revealed that ANXA2 knockdown significantly inhibited osteoblast proliferation and promoted cell apoptosis. RNA-seq analysis demonstrated that ANXA2 knockdown altered the expression levels and alternative splicing of genes associated with cell differentiation, apoptosis, proliferation, and cell cycle regulation. Furthermore, iRIP-seq data indicated that ANXA2 broadly binds RNAs of multiple genes, preferentially recognizing consensus AU-rich motifs within coding regions. Overlap analysis showed that ANXA2 associates with RNAs of genes involved in cell differentiation, apoptosis, proliferation, and cell cycle, and that knockdown-associated changes in expression or splicing occur in a subset of these genes. Additionally, CO-IP/MS experiments showed that ANXA2 extensively associates with RNA-binding proteins in osteoblasts. Collectively, our multi-omics study identifies extensive associations between ANXA2 and transcriptomic/splicing changes in osteoblasts, and shows that ANXA2 broadly associates with RNAs and co-purifies with multiple RNA-binding proteins. These findings provide a comprehensive resource for understanding ANXA2’s potential molecular functions in osteoblasts and generate testable hypotheses for future mechanistic studies.