Abstract: [Objective] To investigate the regulatory effect of homeobox A10（HOXA10） transcription factor on the balance of osteogenic and adipogenic differentiation of sheep bone marrow mesenchymal stem cells （BMSCs） and its molecular mechanism. [Methods] The dynamic expression of HOXA10 mRNA in sheep BMSCs during osteogenic and adipogenic induction was detected by qRT-PCR. The HOXA10 knockdown vector was constructed, and the effects of HOXA10 knockdown on osteogenic and adipogenic differentiation phenotypes were evaluated by Alizarin Red S staining and Oil Red O staining. The effects of HOXA10 knockdown on the expression of osteogenic and adipogenic marker genes were detected by qRT-PCR and Western blot. Combined with JASPAR database analysis, AlphaFold 3 structure prediction and dual-luciferase reporter assays, the regulatory mechanism of HOXA10 on LPL and FABP4 promoters was explored. [Results] ①The relative expression of HOXA10 mRNA initially increased and then decreased in osteogenic induction, and continued to decrease from the second day in adipogenic induction. ②After HOXA10 knockdown, the expression of HOXA10 mRNA and HOXA10 protein were extremely significantly decreased （P<0.01）. ③In terms of osteogenic differentiation, HOXA10 knockdown extremely significantly reduced the mineralization level （P<0.01）, and the mRNA expression of osteogenic marker genes RUNX2 （P<0.01）, ALPL （P<0.05） and OPN （P<0.05） and the protein expression of RUNX2 and ALPL were extremely significantly down-regulated （P<0.01）. In terms of adipogenic differentiation, HOXA10 knockdown extremely significantly increased lipid accumulation （P<0.01）, and the mRNA expression of adipogenic marker genes PPARG （P<0.05）, LPL （P<0.01） and FABP4 （P<0.01） and the protein expression of LPL and FABP4 were extremely significantly up-regulated （P<0.01）. ④The JASPAR database predicted three and six potential HOXA10 binding sites in the LPL and FABP4 promoter regions, respectively. AlphaFold 3 modeling showed that HOXA10 homeodomain could form stable binding interfaces with LPL PB1 and FABP4 PB3. The dual-luciferase reporter assays confirmed that the LPL and FABP4 promoter activities were extremely significantly increased after HOXA10 knockdown （P<0.01）. [Conclusion] HOXA10 may regulate the balance of osteogenic and adipogenic differentiation of sheep BMSCs by inhibiting the transcriptional activity of LPL and FABP4 promoters. HOXA10 knockdown can inhibit osteogenesis and promote adipogenesis, which provides a theoretical basis for the study of mesenchymal stem cell-related disease mechanism, sheep biomedical application and sheep germplasm improvement.

Key words: sheep, HOXA10 gene, bone marrow mesenchymal stem cells, osteogenic differentiation, adipogenic differentiation