Myotube-derived exosomal miR-21 regulates the proliferation and adipogenic differentiation of ovine subcutaneous preadipocytes.

Skeletal muscle-derived signals contribute to intercellular communication with adipose tissue and may influence adipose tissue development; however, the role of exosomes in muscle-adipose crosstalk in sheep remains unclear. This study investigated whether myotube-derived exosomes regulate ovine preadipocyte proliferation and adipogenic differentiation through microRNA-mediated mechanisms. Myotube-preadipocyte interactions were examined using transwell co-culture, exosome isolation and characterization, small RNA sequencing, gain and loss function assays, dual-luciferase assays, and rescue experiments. Myotube co-culture promoted preadipocyte proliferation and suppressed adipogenic differentiation, as indicated by an increased proportion of 5-ethynyl-2'-deoxyuridine (EdU) positive cells and proliferation related markers (P < 0.05) and reduced lipid accumulation and adipogenic markers (P < 0.05). Myotube-derived exosomes, which had a mean diameter of approximately 105 nm and expressed cluster of differentiation 9 (CD9), cluster of differentiation 63 (CD63), and tumor susceptibility gene 101 (TSG101), were internalized by preadipocytes and reproduced these effects (P < 0.05). Small RNA sequencing identified microRNA-21 (miR-21) as the most abundant exosomal miRNA, accounting for approximately 20% of miRNA-mapped reads. Overexpression of miR-21 promoted preadipocyte proliferation and suppressed adipogenic differentiation, whereas miR-21 inhibition produced the opposite responses (P < 0.05). Dual-luciferase assays confirmed programmed cell death protein 4 (PDCD4) and nuclear factor I A (NFIA) as direct targets of miR-21. Rescue experiments showed that PDCD4 and NFIA overexpression attenuated the proliferative and anti-adipogenic effects of miR-21, respectively. Collectively, myotube-derived exosomal miR-21 promotes ovine preadipocyte proliferation by regulating PDCD4 and suppresses adipogenic differentiation through NFIA. These findings reveal an exosome-mediated mechanism underlying muscle-adipose crosstalk in sheep.