MicroRNA‐Seq Analysis Reveals a Critical Role for miR‐222 in Bovine Intramuscular Adipogenesis via the DDIT4/mTORC1 Axis

ABSTRACT Intramuscular fat (IMF) is a key determinant of beef quality, particularly with respect to tenderness, flavor, and juiciness. Increasing evidence indicates that microRNAs (miRNAs) function as important post‐transcriptional regulators in adipogenesis. In this study, microRNA sequencing was performed on IMF tissue obtained from beef samples with distinct marbling scores to characterize miRNA expression profiles. Among the differentially expressed miRNAs, miR‐222 was also significantly regulated during bovine intramuscular preadipocyte differentiation and was therefore selected for further functional and mechanistic investigation. Functional analyses demonstrated that miR‐222 promotes lipid droplet accumulation and triacylglycerol (TAG) synthesis. Moreover, miR‐222 increased the mRNA and protein expression levels of adipogenic marker genes, including fatty acid synthase ( FASN ) and peroxisome proliferator‐activated receptor gamma ( PPARγ ). DNA damage induced transcript 4 ( DDIT4 ) was identified as a direct target of miR‐222. Knockdown of DDIT4 significantly enhanced lipid droplet accumulation and TAG synthesis. Notably, the inhibitory effect of miR‐222 knockdown on adipogenic differentiation was reversed by treatment with the mammalian target of rapamycin complex 1 (mTORC1) agonist MHY1485. In summary, our findings demonstrate that miR‐222 promotes bovine intramuscular adipogenesis by directly targeting DDIT4, thereby activating mTORC1‐mediated lipid synthesis. These results provide new insights into the molecular mechanisms underlying IMF deposition in beef cattle and offer a theoretical basis for genetic improvement strategies.