Excavation and characterization of key circRNAs for milk fat percentage in Holstein cattle

Milk fat percentage is one of the significant indicators governing the price and quality of milk and is regulated by a variety of non-coding RNAs. We used RNA sequencing (RNA-seq) techniques and bioinformatics approaches to explore potential candidate circular RNAs (circRNAs) regulating milk fat metabolism. After analysis, compared with low milk fat percentage (LMF) cows, 309 circRNAs were significantly differentially expressed in high milk fat percentage (HMF) cows. Functional enrichment and pathway analysis revealed that the main functions of the parental genes of differentially expressed circRNAs (DE-circRNAs) were related to lipid metabolism. We selected four circRNAs (Novel_circ_0000856, Novel_circ_0011157, novel_circ_0011944, and Novel_circ_0018279) derived from parental genes related to lipid metabolism as key candidate DE-circRNAs. Their head-to-tail splicing was demonstrated by linear RNase R digestion experiments and Sanger sequencing. However, the tissue expression profiles showed that only Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 were expressed with high abundance in breast tissue. Based on the subcellular localization found that Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 mainly function as competitive endogenous RNAs (ceRNAs) in the cytoplasm. Therefore, we constructed their ceRNA regulatory networks, and the five hub target genes (CSF1, TET2, VDR, CD34, and MECP2) in ceRNAs were obtained by CytoHubba and MCODE plugins in Cytoscape, as well as tissue expression profiles analysis of target genes. These genes play a key role as important target genes in lipid metabolism, energy metabolism, and cellular autophagy. The Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 regulate the expression of hub target genes through interaction with miRNAs and constitute key regulatory networks that may be involved in milk fat metabolism. The circRNAs obtained in this study may act as miRNA sponges and thus influence mammary gland development and lipid metabolism in cows, which improves our understanding of the role of circRNAs in cow lactation.Milk is an important food source, consisting of a complex mixture of lipids, proteins, carbohydrates, and other factors, of which milk fat not only affects the flavor and nutritional value of milk but also plays an important role in the metabolism of nutrients during human growth and development. To dig for potential circular RNAs (circRNAs) and their key regulatory networks that regulate milk fat, we used RNA sequencing (RNA-seq) to identify 309 circRNAs that are differentially expressed between the mammary epithelial cells (MECs) of cows with high and low milk fat percentage. We screened key circRNAs and their circRNA-miRNA-mRNA regulatory networks affecting milk fat by bioinformatic methods. It provides a new way to study lactation, milk quality, and breed improvement in dairy cows.