Study on the Role of FHL2 in Chicken: Promoting Bone Growth and Inhibiting Fat Formation via Regulating BMSC Differentiation Balance

In this study, the objective was to identify the key regulators of the osteogenic–adipogenic differentiation equilibrium of chicken bone marrow mesenchymal stem cells (BMSCs), with the expectation of developing marker genes that promote skeletal development and inhibit excessive fat deposition. BMSCs were isolated and phenotypically characterized. qRT-PCR showed FHL2 increased during osteogenic differentiation. Transfection with FHL2 overexpression plasmid or siRNAs was done. FHL2 overexpression increased osteogenic marker mRNA (RUNX2, ALP, Osterix, and OCN) levels, reduced lipid deposition-related gene (C/EBPα, C/EBPβ) expression, and increased mineralized area and nodules. FHL2 knockdown had the opposite effect. FHL2 overexpression also reduced apoptosis and proliferation-related transcripts, decreased apoptosis rate, and reduced proliferation-related transcripts and altered cell-cycle distribution. After FHL2 knockdown, RNA sequencing identified 81 differentially expressed genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed changes in various processes and pathways. Enrichment analysis indicated that the differentially expressed genes were involved in apoptosis, cytokine–cytokine receptor interaction, Th17 cell differentiation, JAK-STAT signaling, cAMP signaling, steroid hormone biosynthesis, and retinol metabolism. These findings indicate that FHL2 promotes osteogenic differentiation and suppresses adipogenic differentiation in chicken BMSCs in vitro and may act through multiple downstream pathways.