Abstract
Background：In the bovine mammary gland, de novo fatty acid synthesis is a critical process for milk fat production, in which acetyl-CoA synthetase 2 (ACSS2) serves as a key enzyme by converting acetate into acetyl-CoA. This metabolic pathway is intricately regulated by non-coding RNAs, particularly through the competitive endogenous RNA (ceRNA) mechanism.Purpose: To elucidate the regulatory role and molecular mechanism of the circRNA-02213/miR-328/ACSS2 axis in the lipid metabolism of bovine mammary epithelial cells (BMECs). Methods: Bioinformatic prediction and dual-luciferase reporter assays were employed to verify the targeting interactions among circRNA-02213, miR-328, and ACSS2. In BMECs, qRT-PCR, Western blot, triglyceride/cholesterol quantification, Oil Red O staining, and cell proliferation assays were used to evaluate the effects of this axis on key lipid-metabolic indices and cellular phenotypes. Results: circRNA-02213 functioned as a molecular “sponge” that sequestered miR-328, thereby upregulating ACSS2 expression. Functionally, circRNA-02213 overexpression markedly promoted triglyceride and cholesterol synthesis, lipid droplet accumulation, and BMEC proliferation; whereas miR-328 exerted significant inhibitory effects on these lipid-metabolic processes and cell proliferation. Conclusion: This study demonstrates that circRNA-02213 acts as a ceRNA to relieve miR-328-mediated repression of ACSS2, constituting a critical network that regulates milk fat synthesis and metabolism. The circRNA-02213/miR-328/ACSS2 axis represents a potential molecular target for improving milk lipid quality in ruminants.