Assessment of microRNA profiles in small extracellular vesicles isolated from bovine colostrum with different immunoglobulin G concentrations

Abstract

The consumption of bovine colostrum by newborn calves during the first days of life is essential to ensure the transfer of passive immunity. In addition to critical IgG, colostrum also contains non-IgG biomolecules, including microRNA (miRNA). The present study investigated the profiles of miRNA in small extracellular vesicles (sEV) isolated from bovine colostrum with high (256.5 ± 5.7 mg/mL, mean ± standard deviation, n = 4) and low (62.8 ± 3.6 mg/mL, n = 4) concentrations of IgG. Different combination of sEV extraction methods and bioinformatic pipelines (miRDeep2 and sRNAbench) for miRNA analysis were evaluated. Results showed that miRCURY exosome Cell/Urine/CSF and miRNeasy Mini kits yielded the highest RNA concentration. The miRNA-seq data analysis showed miRDeep2 yielded more comprehensive miRNAome compared with sRNAbench (527 versus 392 unique miRNA), whereas 389 shared miRNA were identified using both approaches. The profiles of top 50 miRNA were the same using both approaches, and their abundance contributed to 91.7% and 94.3% of total abundance of miRNA using miRDeep2 and sRNAbennch, respectively. These core miRNA were predicted to target 2,655 genes, which regulate 78 KEGG (Kyoto Encyclopedia of Genes and Genomes) level-3 pathways including PI3K-Akt and MAPK signaling pathway, axon guidance, and focal adhesion. The expression profiles of sEV-associated miRNA were similar between high- and low-IgG colostrum samples, despite the fact that the abundance of miR-27a-3p was higher in colostrum with high concentrations of IgG. In conclusion, a core miRNAome in bovine colostrum may play a role in regulating health and developmental stages in neonatal calves, independent of IgG concentration.

Summary: A total of 389 microRNA (miRNA) were identified in small extracellular vesicles (sEV) isolated from bovine colostrum, with the top 50 miRNA contributing to more than 90% of total abundance and predicted to target 2,655 genes associated with cellular processes, environmental information processing, and organismal systems. The expression profiles of sEV-associated miRNA in bovine colostrum was independent of the concentrations of immunoglobulin G.

Figure 1

(A) Abundance of the top 50 small extracellular vesicle (sEV)-associated microRNA (miRNA) in the 8 bovine colostrum samples identified by 2 different bioinformatic pipelines: miRDeep2 (purple bar; Friedländer et al., 2012) and sRNAbench (blue bar; Aparicio-Puerta et al., 2019). (B) Main Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (generated using the Database for Annotation, Visualization, and Integrated Discovery; DAVID) regulated by the predicted target genes [generated using TargetScan (http://www.targetscan.org/vert_80/), probability of preferentially conserved targeting (P_CT) score was set to 0.8] of the top 50 sEV-associated miRNA in the 8 bovine colostrum samples. CPM = counts per million reads.

Figure 2

Profiles of small extracellular vesicle (sEV)-associated microRNA (miRNA) in bovine colostrum with different IgG concentrations identified by miRDeep2 (Friedländer et al., 2012). (A) Difference in concentrations of IgG between 2 groups of samples. (B) The principal component (PC) analysis plot of the miRNA profiles in high- and low-IgG colostrum. (C) Differential abundance of miR-27a-3p in high- and low-IgG colostrum samples (P = 0.028). The differential expression analysis was conducted using DESeq2 (Love et al., 2014). Significant differences were declared at Benjamini-Hochberg adjusted P < 0.05. CPM = counts per million reads.