Bovine Preadipocyte-Derived Exosomal miR-26a Promotes Intramuscular Adipogenesis by Directly Targeting GSK3B

Intramuscular fat (IMF) deposition is a critical determinant of beef quality, yet the regulatory role of preadipocyte-derived exosomes remains unclear. Exosomes (HC-Exo and CRSC-Exo) were isolated from preadipocytes of Haoyue Beef Cattle (HC) and Chinese Red Steppe Cattle (CRSC), breeds with distinct IMF contents. HC-Exo significantly enhanced preadipocyte proliferation and differentiation, increasing lipid droplet formation and triglyceride synthesis, alongside elevated expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer binding protein alpha (C/EBPα). MiRNA sequencing identified 580 differentially expressed miRNAs, with miR-26a twice as abundant in HC-Exo versus CRSC-Exo. Integrated bioinformatics analyses, including KEGG, GO, and dual-luciferase reporter assays, identified glycogen synthase kinase-3B (GSK3B) as a direct target of miR-26a. Functional validation demonstrated that miR-26a mimics enhanced adipogenesis by suppressing GSK3B expression, while GSK3B overexpression inhibited differentiation. These findings establish exosomal miR-26a as a novel regulator of intramuscular adipogenesis, providing molecular insights for improving beef quality.