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

Abstract

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, 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.

Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages

“…The experimental methodology in this section is also consistent with our previous research methodology [ 66 ]. TRIzol reagent was used to extract the total RNA from tissues of the heart, liver, kidney, uterus, ovaries, small intestine, and mammary gland.…”

“…The sequencing in the present study belongs to the same batch as that in a recent study [ 66 ] and is, therefore, methodologically identical. Total RNA was extracted from cow milk MECs using the TRIzol method.…”

“…Identification of circRNAs and differential expression analysis were performed according to our previously described methods [ 66 ]. The circRNA was detected and identified using find_circ [ 67 ] and CIRI2 [ 68 ].…”

“…Subsequently, the head-to-tail splice sites of the circRNAs were identified through Sanger sequencing. The circular structure was verified using our previous verification method [ 66 ]. Table S7 lists the primer sequences used in the present study.…”

Understanding Circular RNAs in Health, Welfare, and Productive Traits of Cattle, Goats, and Sheep

“…Finally, the researchers identified five crucial ceRNA networks that potentially regulate mammary gland development, lactation, and milk fat in dairy cows. These networks involve novel circ0011157/bta-miR-1291/ CSF1 , novel circ0011157/bta-miR-1777b/ VDR , novel circ0011157/bta-miR-1777b/ MECP2 , novel circ0000856/bta-miR-29d-3p/ TET2 , and novel circ0011944/bta-miR-12033/ CD34 [ 32 ].…”