A nucleus-associated miR-661-C/EBPα-PPARγ regulatory axis skews BMSC lineage commitment in SONFH.

Steroid-induced osteonecrosis of the femoral head (SONFH) is characterized by marrow adiposity, trabecular deterioration, and impaired bone repair, but the progenitor-level regulatory mechanisms underlying this process remain incompletely defined. Here, we investigated whether dysregulated bone marrow stromal cell (BMSC) lineage commitment contributes to SONFH progression and identified a nucleus-associated miRNA mechanism that promotes adipogenic skew. Human femoral heads from patients with femoral neck fracture or SONFH showed increased empty osteocytic lacunae, trabecular rarefaction, and Perilipin-1-positive marrow adiposity. Patient-derived SONFH BMSCs displayed enhanced adipogenic potential and impaired osteogenic differentiation, accompanied by increased C/EBPα/PPARγ and reduced RUNX2/OCN expression. Cross-source miRNA prioritization and validation identified miR-661 as an upregulated candidate associated with this lineage imbalance. miR-661 overexpression promoted lipid accumulation, suppressed mineral deposition, and activated a pro-adipogenic transcriptional program. Mechanistically, miR-661 showed nuclear enrichment and did not induce a detectable change in β-catenin expression or nuclear localization, directing our focus to the C/EBPα-PPARγ axis. ChIP-qPCR, promoter DNA pull-down, dual-luciferase reporter assays, and C/EBPα RIP-qPCR showed that miR-661 was associated with increased C/EBPα occupancy at the PPARG promoter. Importantly, C/EBPα knockdown attenuated miR-661-induced PPARG, FABP4, and adipogenic activation, whereas C/EBPα rescue restored these effects. Nuclear AGO2-RIP-qPCR further demonstrated enrichment of miR-661 in nuclear AGO2-containing complexes, and AGO2 ChIP-qPCR showed miR-661-dependent AGO2 occupancy at the PPARG promoter, which was reduced by miR-661 inhibition and lost with a seed-mutant miR-661 mimic. In vivo, local antagomiR-661 delivery alleviated steroid-induced osteonecrosis-like bone loss and marrow adiposity. These findings identify miR-661 as a nuclear, AGO2-associated regulator of the C/EBPα-PPARγ axis that drives BMSC adipogenic lineage bias in SONFH.