Identification and Characterization of Circular RNAs in Mammary Tissue from Holstein Cows at Early Lactation and Non-Lactation

Abstract

In this study, circular RNAs (circRNAs) from Holstein cow mammary tissues were identified and compared between early lactation and non-lactation. After analysis, 10,684 circRNAs were identified, ranging from 48 to 99,406 bp, and the average size was 882 bp. The circRNAs were mainly distributed on chromosomes 1 to 11, and 89.89% of the circRNAs belonged to sense-overlapping circRNA. The exons contained with circRNAs ranged from 1 to 47 and were concentrated from 1 to 5. Compared with the non-lactating cows, 87 circRNAs were significantly differentially expressed in the peak lactation cows. There were 68 upregulated circRNAs and 19 downregulated circRNAs. Enrichment analysis of circRNAs showed that GO analysis mainly focused on immune response, triglyceride transport, T cell receptor signaling pathway, etc. Pathway analysis mainly focused on cytokine-cytokine receptor interaction, T helper 17 cell differentiation, fatty acid biosynthesis, the JAK-STAT signaling pathway, etc. Specific primers were designed for two proximal ends of the circRNA junction sites to allow for PCR validation of four randomly selected circRNAs and carry out circRNA-miRNA interaction research. This study revealed the expression profile and characteristics of circRNAs in mammary tissue from Holstein cows at early lactation and non-lactation, thus providing rich information for the study of circRNA functions and mechanisms, as well as potential candidate miRNA genes for studying lactation in Holstein cows.

Keywords: Holstein cows; RNA sequencing; circular RNA; mammary tissue.

Figure A1

Network regulation of mRNA–circRNA–miRNA.

Figure 1

CircRNA numbers predicted in each sample. The vertical axis is the number of circRNAs; the horizontal axis shows the individual samples from cows on postpartum days 30 or 315; the numbers above each bar refer to the number of circRNAs predicted in each sample; the Uniq_circRNA_numbers refer to the number of circRNAs specifically predicted in each sample compared to other samples in the project.

Figure 2

Identification, characterization and chromosomal distribution of circRNAs. (A) The number of circRNAs per chromosome; (B) the length distribution density of circRNAs); (C) the number of exons by circRNAs; (D) statistical diagram of circRNA shear signal, with the number of circRNAs on the vertical axis and the type of shear signal on the horizontal axis (specific data are presented in the Supplementary Materials, Table S1); (E) the vertical axis is the number of circRNAs and the horizontal axis shows the CG content of circRNAs; (F) percentage of different types of circRNAs.

Figure 3

Differentially expressed circRNAs in mammary tissue from Holstein cows at early lactation and non-lactation. (A) The X-axis is the mean expression of all samples used for comparison after standardization. The Y-axis is the Log₂ fold change. The red highlights are significantly differently expressed circRNAs. (B) Gray and blue circRNAs with non-significant differences; red and green circRNAs with upregulated and downregulated significant differences, respectively. The X-axis is the log₂ fold change and the Y-axis is the log₁₀ p-value.

Figure 4

Top 30 categories of GO analysis. The basic information for each node, i.e., the GO ID and GO term, is displayed in the corresponding graph. The X-axis is the GO entry name and the Y-axis is the log₁₀ p-value.

Figure 5

Top 20 categories of KEGG pathway analysis. KEGG enrichment top 20 bubble diagram, where the X-axis is the Enrichment Score. The larger the bubble, the more circRNAs the item contains, and the color of the bubble changes from purple to blue to green to red. The smaller the Enrichment p-value, the greater the significance.

Figure 6

RT-PCR validation of the presence of circRNAs in cow mammary gland tissue. (A) Reverse transcriptase-polymerase chain reaction (RT-PCR) amplimers derived from the circular RNAs using divergent primers for cows’ mammary gland RNA (M: marker); (B) Head-to-tail splice junctions for the circRNAs were confirmed by DNA sequencing and are marked with a red arrow on the DNA sequence chromatograms.

Figure 7

Four-network regulation of mRNA–circRNA–miRNA.