Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (24): 5274-5284.doi: 10.3864/j.issn.0578-1752.2025.24.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

FoxO1 Inhibits the Proliferation and Differentiation of Bovine Myoblasts and Adipocytes Through TGF-β/SMAD-TGFBI Pathway

JIANG Chao1,2(), ZHANG JiuPan3, SONG YaPing1,2, JIAO RuoPu1,2, YANG DongMei1,2, GONG HongFang1,2, MA YiLun1,2, MA Yun1,2, WEI DaWei1,2,*()   

  1. 1 College of Animal Science and Technology, Ningxia University, Yinchuan 750021
    2 Key Laboratory of Ruminant Molecular Cell Breeding, Ningxia Hui Autonomous Region, Yinchuan 750021
    3 Institute of Animal Science, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750021
  • Received:2025-06-26 Accepted:2025-08-03 Online:2025-12-22 Published:2025-12-22
  • Contact: WEI DaWei

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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

Table 1

Synthesis information of siRNA"

名称 Name 序列Sequence (5′→3′)
si-FoxO1 正链Sense CACACAGUGUCAAGACAATT
反链Anti-Sense UUGUCUUGACACUGUGUGGTT
si-NC 正链Sense UUCUCCGAACGUGUCACGUTT
反链Anti-Sense ACGUGACACGUUCGGAGAATT

Table 2

Antibody information"

抗体名称 Antibody name 种属 Source 大小 Size (kDa) 稀释比例 Dilution ratio 制造商 Manufacturer
SMAD2 兔 Rabbit 58 1:1000 Abways
p-SMAD2 兔 Rabbit 58 1:1000 Abways
GAPDH 兔 Rabbit 36 1:1000 Abways

Fig. 1

Screening of common KEGG signaling pathway A: KEGG analysis of bovine myoblasts and preadipocytes that interfere with FoxO1 and induce differentiation; B: Significantly enriched KEGG signaling pathway in bovine myoblasts; C: Significantly enriched KEGG signaling pathway in bovine adipocytes"

Fig. 2

Screening of common differential genes A: DEGs of bovine myoblasts and adipocytes; B: Common DEGs screening"

Fig. 3

Expression of TGF-β signaling pathway marker protein in bovine myoblasts and preadipocytes after interference with FoxO1 A: Expression levels of SMAD2 and p-SMAD2 proteins in bovine skeletal muscle cells after interference with FoxO1; B: p-SMAD2/ total SMAD2 in bovine myoblasts; C: Expression levels of SMAD2 and p-SMAD2 proteins in bovine preadipocytes after interference with FoxO1; D: p-SMAD2/ total SMAD2 in bovine preadipocyte"

Fig. 4

Selection of optimal concentrations of activators and inhibitors of TGF-β signaling pathway A and B: Expression of SMAD2 and p-SMAD2 proteins in bovine myoblasts and preadipocytes treated with DMSO, activator and inhibitor with different concentrations"

Fig. 5

EdU detection of proliferation ability of bovine myoblasts A: EdU staining; B: Relative cell proliferation rate"

Fig. 6

EdU detection of proliferation ability of bovine preadipocytes A: EdU staining; B: Relative cell proliferation rate"

Fig. 7

Flow cell cycle detection of bovine myoblasts and preadipocytes A and B: Cell cycle detection of bovine myoblasts; C and D: Cell cycle detection of bovine preadipocytes"

Fig. 8

Detection of myotube formation by immunofluorescence staining"

Fig. 9

Detection of differentiation ability of bovine preadipocytes A: Oil red O staining; B: Relative area of lipid droplets; C: Bodipy staining"

Fig. 10

Prediction and verification of targeting relationship between FoxO1 and TGFBI A: The target binding site of FoxO1 and TGFBI promoter region; B: Double luciferase activity detection"

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