Introduction and objective:
MicroRNAs (miRNAs) regulate osteogenic differentiation in osteoporotic fractures (OF). The aim of the study is to investigate the diagnostic value and regulatory role of miR-4666a-3p in OF.

Material and methods:
The study is based on clinical samples (89 healthy controls, 79 OP patients, 83 OF patients) and hFOB1.19 cell experiments. RT-qPCR analyzed miR-4666a-3p expression, and logistic regression identified OF risk factors. ELISA measured RANKL and OPG concentrations. In vitro, osteoblast differentiation was induced to assess ALP activity, Runx2, Osteocalcin, Col1a1, cell viability (CCK-8), and apoptosis (flow cytometry). Target genes were bioinformatically predicted and confirmed via dual-luciferase assay. Rescue experiments explored the role of GSK3B in miR-4666a-3p-mediated osteogenic regulation. All experiments were repeated at least three times.

Results:
Downregulation of miR-4666a-3p could distinguish healthy controls and OP patients, further differentiate between OP and OF patients, and may predict the risk of OF occurrence. During osteoblast differentiation, miR-4666a-3p expression increased, ALP activity elevated, and key marker levels upregulated. Overexpression of miR-4666a-3p promoted osteoblast differentiation and activity, and reduced apoptosis rate, whereas inhibiting miR-4666a-3p had the opposite effect. MiR-4666a-3p negatively regulated the downstream target GSK3B. Overexpression of GSK3B reversed the osteogenic effects of miR-4666a-3p.

Conclusions:
Downregulation of miR-4666a-3p may predict OF risk. Its upregulation promoted osteoblast differentiation by targeting GSK3B, potentially facilitating fracture healing.