TGF-β1-regulation of bone formation via the miR-3064-3p/HDAC4/Runx2 axis: In vitro and in vivo studies

Transforming growth factor-beta1 (TGF-β1) regulates bone formation through Runx2, a bone transcription factor, and Runx2 activity is regulated by several coactivators and corepressors. Histone deacetylase 4 (HDAC4) functions as a transcriptional corepressor, and it downregulates Runx2 activity in osteoblastic cells. Emerging evidence suggests that non-coding RNAs, particularly microRNAs (miRNAs), significantly influence normal and abnormal bone homeostasis. In this study, we identified miR-3064-3p, which targets HDAC4 in osteoblasts. Our findings demonstrated that TGF-β1 upregulates miR-3064-3p, which in turn downregulates HDAC4, leading to increased acetylation of Runx2 in osteoblastic cells. Conversely, inhibition of miR-3064-3p restored HDAC4 levels and reduced Runx2 acetylation. Inhibition of miR-3064-3p also decreased the expression of osteogenic transcription factors in differentiating osteoblasts. miR-3064-3p directly targeted the 3' untranslated region of HDAC4 in osteoblasts. In vivo analyses using a rat femoral defect model demonstrated that TGF-β1 significantly enhances bone regeneration, and the miR-3064-3p inhibitor markedly impaired this regenerative process, indicating its essential role in mediating TGF-β1-driven bone regeneration. Collectively, these findings highlight the pivotal role of the TGF-β1/miR-3064-3p/HDAC4/Runx2 axis as a positive regulator of bone formation.