Inhibition of miR-33b-5p promotes osteoblast differentiation by regulating ABCA1 in cellular and animal models of osteoporosis

INTRODUCTION: Osteoporosis is a major health risk for postmenopausal women and is primarily driven by estrogen decline-induced bone loss. As critical post-transcriptional regulators of gene expression, dysregulated microRNAs (miRs) participate in the pathogenesis of osteoporosis by disrupting key signaling pathways that govern bone homeostasis and remodeling. This study investigated the functional role of the miR-33b-5p/ABCA1 axis in cellular and murine models of osteoporosis. MATERIAL AND METHODS: Alizarin Red S staining and alkaline phosphatase (ALP) activity assays were performed to detect osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). ABCA1 mRNA and protein levels, as well as osteogenic proteins such as RUNX2, OPN, and OCN, were quantified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. Dexamethasone (DEX)-stimulated BMSCs were employed as an in vitro osteoporosis model. To overexpress ABCA1 in BMSCs, the cells were transfected with pcDNA3.1 vectors carrying the full-length ABCA1 coding sequence. ABCA1 silencing was accomplished using short hairpin RNA targeting ABCA1 (sh-ABCA1), whereas miR-33b-5p inhibition was achieved using a specific miR-33b-5p inhibitor. The direct interaction between miR-33b-5p and its downstream target ABCA1 was validated using luciferase reporter assays. After an ovariectomized (OVX) mouse model was established, micro-CT was performed to detect bone density by assessing trabecular thickness and trabecular number in femurs. RESULTS: ABCA1 expression increased during osteogenic differentiation of BMSCs, suggesting an essential role in this process. In DEX-treated BMSCs, the ABCA1 level was downregulated. ABCA1 overexpression promoted osteoblast differentiation, as evidenced by increased mineralized nodule formation, upregulation of key osteogenic proteins, and elevated ALP activity. MiR-33b-5p directly interacted with the 3'UTR of ABCA1, with the binding region conserved across multiple species. MiR-33b-5p inhibitors promoted osteogenic differentiation, and this effect was abolished upon ABCA1 depletion via sh-ABCA1. Animal experiments indicated that miR-33b-5p inhibition promoted trabecular bone formation in the femurs of OVX mice, including increased trabecular number and thickness, along with elevated expression of osteoblastic markers. A significant negative correlation between miR-33b-5p and ABCA1 expression levels was observed in bone tissues of experimental mice. CONCLUSIONS: . Inhibition of miR-33b-5p promotes osteoblastic differentiation by regulating ABCA1 in experimental osteoporosis models.