Construction of the ceRNA Regulatory Network Associated with Milk Fat Metabolism

Milk fat composition and content are crucial indicators that influence the taste, flavor and nutritional value of milk, and they are regulated by various non-coding RNAs. To enhance milk quality, it is essential to investigate the regulatory mechanisms underlying milk fat metabolism. This study analyzed significantly up-regulated circ_0009058 and circ_0004021 and significantly down-regulated circ_0011934 and circ_0008056 in bovine mammary epithelial cells (BMECs) with high and low milk fat percentages (MFP). RNase R digestion assays and Sanger sequencing were conducted to confirm their presence and high expression in the mammary tissue of dairy cows. Subcellular localization indicated that these RNA may exhibit regulatory functions through the competing endogenous RNA (ceRNA) network. Consequently, a ceRNA regulatory network was constructed, identifying six hub target genes in the ceRNA network (VAV1, PTPN6, PIK3R1, RHOA, ERBB3, PIK3CG) using the CytoHubba and MCODE plugins in Cytoscape (version 3.9). Quantitative real-time PCR (RT-qPCR) and dual luciferase reporter gene assays validated the existence of the interactive regulatory networks circ_0004021/bta-miR-541/PTPN6, circ_0008056/bta-miR-2309/ERBB3, and bta-miR-10175-3p/RHOA in BMECs. Functional validation of miRNAs within the ceRNA network demonstrated that bta-miR-541 and bta-miR-10175-3p reduced triglyceride and cholesterol levels while inhibiting lipid droplet secretion in BMECs. Conversely, the function of bta-miR-2309 was found to be opposite to that of bta-miR-541 and bta-miR-10175-3p. This study identified several critical candidate ceRNA networks involved in fatty acid metabolism in dairy cows through data analysis and a series of experiments. These functional and mechanistic studies provide a theoretical foundation for improving milk quality.