The 3’UTR protects bone against menopause-induced osteoporosis via regulating CHD9 expression

Abstract Osteoporosis is characterized by low bone mass, increased bone fragility, and elevated fracture susceptibility, with approximately one-third of postmenopausal women affected by this disease. Estrogen deficiency induces aberrant upregulation of microRNA (miRNA) expression, and the 3’-untranslated region (3’UTR) of mRNAs can bind to and sequester target miRNAs. This study aimed to identify target genes capable of sequestering and inhibiting miRNAs in postmenopausal osteoporosis (PMOP). Candidate differentially expressed microRNAs were screened by analyzing sequencing data from the whole blood and serum of PMOP patients. These candidate microRNAs were validated in the serum of PMOP patients, the serum and bone tissue of ovariectomized (OVX) mice, as well as in mouse osteoblasts and osteoclasts. Luciferase reporter assays were performed to verify the binding sites between the candidate microRNAs and their predicted target genes; argonaute 2 RNA immunoprecipitation (AGO2-RIP) assays were carried out to detect ceRNA binding at the cellular level, and in vivo therapeutic animal experiments were also implemented. We screened and cross-validated miR-144-5p and miR-532-5p, which were found to be highly expressed in the serum of PMOP patients, the serum and bone tissue of OVX mice, and mouse osteoblasts. The common target gene of these two miRNAs is chromodomain helicase DNA-binding protein 9 ( CHD9 ). Luciferase reporter assays confirmed that the p233-239 and p721-727 regions within the CHD9 3’UTR are the specific binding sites for miR-144-5p and miR-532-5p, respectively. In osteoblasts, CHD9 positively regulates the expression of runt-related transcription factor 2 ( RUNX2 ) by binding to the − 450 to -400 region of the RUNX2 promoter. In vivo experiments further demonstrated that the CHD9 3’UTR ameliorates ovariectomy-induced osteoporotic phenotypes. This study demonstrates that the CHD9 3’UTR offers a potential therapeutic strategy for postmenopausal osteoporosis.