Regulatory Role of microRNA of Milk Exosomes in Mastitis of Dairy Cows

Abstract

The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows.

Keywords: cow; exosomes; mastitis; miRNA; milk.

Figure 1

Mean small RNA and microRNA (miRNA) abundance in reads per million (RPM) of the milk samples collected from healthy and mastitic cows.

Figure 2

Venn diagram showing the number of unique or shared differentially expressed miRNAs in milk exosomes sampled from healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). H_ARM, H_SCM, and ARM_SCM refer to the pairwise comparisons between groups (Supplementary Table S1). Venn diagram was created by a web tool available at http://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 14 December 2022.

Figure 3

Venn diagram showing the number of unique or shared KEGG pathways of the target genes significantly enriched in the comparisons between healthy (H) cows, cows with subclinical mastitis (SCM), and cows at risk of mastitis (ARM). H_ARM, H_SCM, and ARM_SCM refer to the pairwise comparisons between groups (Supplementary Table S3). Venn diagram was created by a web tool available at http://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 14 December 2022.

Figure 4

Network analysis of miRNA differentially expressed between healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Figures were generated by miRNet suite [50]. (A) H_ARM comparison: in red are the genes of the NF-KB signaling pathway; in green the genes of the T cell receptor signaling pathway; in yellow the genes of the TNF signaling pathway; in purple the genes of the leukocyte transendothelial migration; and in black the genes of the adherens junction. (B) H_SCM comparison: in red are the genes of the NOD-like signaling pathway; in green the genes of the T cell receptor signaling pathway; in yellow the genes of the TNF signaling pathway; in purple the genes of the leukocyte transendothelial migration; in black the genes of the adherens junction. (Supplementary Table S4 reports the miRNAs in the networks).