关键词: FoxO1, 牛成肌细胞, 前体脂肪细胞, TGF-β信号通路, TGFBI

Abstract:

【Background】The development of skeletal muscle tissue and intramuscular fat tissue directly affects beef quality, and the proliferation and differentiation of myoblasts and preadipocytes play a key role in its growth and development. Previous studies have found that Forkhead box protein O1 (FoxO1) played a transcriptional regulation role in the proliferation and differentiation of bovine myoblasts and preadipocytes, respectively, but the co-regulation mechanism in these two cells was still unclear. 【Objective】The purpose of this study was to explore the key regulatory pathways of FoxO1 in bovine myoblasts and preadipocytes, so as to provide a theoretical basis for clarifying that FoxO1 was a key genetic regulatory factor affecting beef quality. 【Method】Bovine myoblasts and preadipocytes that interfered with FoxO1 and induced differentiation for 4 days were analyzed by transcriptome sequencing, and the significantly enriched KEGG signaling pathway and significantly differentially expressed genes that FoxO1 regulated the differentiation of bovine myoblasts and preadipocytes were screened out. Small interfering RNA (siRNA) was used to inhibit the expression of FoxO1 gene in cells. Western blot (WB) was used to detect the expression level of key signal pathway marker proteins. The effects of key signaling pathways on the relative proliferation rate of bovine myoblasts and preadipocytes were detected by EdU staining. The effects of key signaling pathways on cell cycle distribution of bovine myoblasts and preadipocytes were analyzed by flow cytometry. Immunofluorescence staining was used to detect the effects of key signaling pathways on myotube formation of bovine myoblasts, and oil red O/Bodipy staining was used to detect the effect of key signaling pathways on lipid droplet formation ability of preadipocytes. The interaction between FoxO1 and target gene was verified by double luciferase reporter gene test. 【Result】A total of 11 KEGG signaling pathways and 13 differentially expressed genes were screened by transcriptome analysis, among which TGF-β signaling pathway and TGFBI gene were closely related to myogenesis and lipogenesis, and TGFBI gene was the downstream factor of TGF-β signaling pathway and directly regulated by it. WB detection results showed that interfering with FoxO1 would significantly increase the level of p-SMAD2/total SMAD2 in bovine myoblasts and preadipocytes, indicating that interfering with FoxO1 gene expression would activate TGF-β signaling pathway in bovine myoblasts and preadipocytes. Further detection confirmed that the treatment concentration of TGF-β signal pathway activator and inhibitor in bovine myoblasts and preadipocytes was 5 µmol·L^-1. EdU test showed that activating TGF-β signaling pathway would significantly reduce the relative proliferation rate of bovine myoblasts and preadipocytes (P < 0.01). The results of flow cytometry showed that activating TGF-β signaling pathway would inhibit the G1/S phase transformation of bovine myoblasts and preadipocytes. The results of myotube staining and lipid droplet staining showed that activation of TGF-β signaling pathway inhibited myotube formation of bovine myoblasts and lipid droplet formation of bovine preadipocytes. However, inhibiting TGF-β signaling pathway had the opposite effect. The double luciferase reporter gene test confirmed that FoxO1 could bind to -509—-499 bp and -490—-480 bp regions of TGFBI promoter, and up-regulate its transcription activity. 【Conclusion】FoxO1 could inhibit the proliferation and differentiation of bovine myoblasts and preadipocytes through TGF-β/SMAD-TGFBI pathway.

Key words: FoxO1, bovine myoblasts, preadipocytes, TGF-β signaling pathway, TGFBI