Forkhead box O3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells

1. In the poultry industry, growth performance is important due to its effects on economic value. Much effort has been put forth to achieve introgression of specific genes and DNA markers related to muscle proliferation and differentiation in selective breeding approaches. 2. This study investigated the biological functions of the gene Forkhead box O3 (FOXO3) during myogenic differentiation in chicken myoblast cells. FOXO3 was downregulated in primary chicken myoblast (pCM) cells by the piggyBac transposon-mediated microRNA (miRNA) knock-down (KD) system. 3. The pCM cells that were stably integrated into the FOXO3 KD expression vector showed significant downregulation of FOXO3 protein and mRNA levels. Expression levels of paired box protein Pax7 (Pax7) and target genes such as CCAAT/enhancer binding protein beta and serum response element decreased in FOXO3 KD pCM cells. In addition, in the undifferentiated myoblast stage, there were no significant differences in cell morphology; however, proliferation rate in FOXO3 KD pCM cells was significantly lower during d 4 and 5 of in vitro culture. By contrast, when myotube differentiation was induced, FOXO3 KD pCM cells exhibited rapid initiation of myotube formation, higher expression of myogenin and desmin as myogenic indicators and a further differentiated phenotype than observed in regular pCM cells. 4. These results demonstrated that FOXO3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells. Therefore, the regulation of FOXO3 could be applied to improve muscle differentiation in commercial poultry.