Expression Profile of miRNA from High, Middle, and Low Stress-Responding Sheep during Bacterial Endotoxin Challenge

Abstract

Animals respond to stress by activating a wide array of physiological and behavioral responses that are collectively referred to as the stress response. MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in the regulation of homeostasis. There are many reports demonstrating examples of stress-induced miRNA expression profiles. The aim of this study was to determine the circulatory miRNA profile of variable stress-responding lambs (n = 112) categorized based on their cortisol levels as high (HSR, 336.2 ± 27.9 nmol/L), middle (MSR, 147.3 ±9.5 nmol/L), and low (LSR, 32.1 ± 10.4 nmol/L) stress responders post-LPS challenge (400 ng/kg iv). Blood was collected from the jugular vein at 0 (T0) and 4 h (T4) post-LPS challenge, and miRNAs were isolated from four animals from each group. An array of 84 miRNAs and 6 individual miRNAs were evaluated using qPCR. Among 90 miRNAs, there were 48 differentially expressed (DE) miRNAs (log fold change (FC) > 2 < log FC) in the HSR group, 46 in the MSR group, and 49 in the LSR group compared with T0 (control) samples. In the HSR group, three miRNAs, miR-485-5p, miR-1193-5p, and miR-3957-5p were significantly (p < 0.05) upregulated, while seven miRNAs, miR-376b-3p, miR-376c-3p, miR-411b-5p, miR-376a-3p, miR-376b-3p, miR-376c-3p, and miR-381-3p, were downregulated (p < 0.05) as compared to the LSR and MSR groups. Functional analysis of DE miRNAs revealed their roles in Ras and MAPK signaling, cytokine signaling, the adaptive immune system, and transcription pathways in the HSR phenotype, implicating a hyper-induced acute-phase response. In contrast, in the LSR group, enriched pathways included glucagon signaling metabolic regulation, the transportation of amino acids and ions, and the integration of energy metabolism. Taken together, these results indicate variation in the acute-phase response to an immune stress challenge, and these miRNAs are implicated in regulating responses within cortisol-based phenotypes.

Keywords: biomarker; lipopolysaccharide; microRNA; stress responder.

Figure 1

Basal (T0) and 4 h (T4) concentrations of serum cortisol, IL-6, and IL-10 in sheep before and after LPS (400 ng/kg) i.v. administration. Animals were classified as high (HSR), middle (MSR), or low (LSR) based on their T4 cortisol concentrations. Values are presented as means ± SEM. Significant differences are shown by asterisks (** p ≤ 0.01 and *** p ≤ 0.001) compared with the T0 (basal) levels.

Figure 2

Fold change in expression of candidate ovine microRNAs (miR-145, miR-200b, miR-233, miR-1246, miR-29b, miR-31, and miR-130) before (T0) and 4 h (T4) after LPS (400 ng/kg) i.v. administration. The asterisks (* p ≤ 0.05 and ** p ≤ 0.01) indicate significant differences between T0 and T4.

Figure 3

Volcano plots of differentially expressed miRNAs 4 h post-LPS (400 ng/kg) i.v. administration from variable stress-responding groups of sheep: (a) HSR vs. LSR, (b) HSR vs. MSR, and (c) MSR vs. LSR. (d) Venn diagram of genes commonly upregulated and downregulated in all groups. Number digits are numbers of miRNAs that are up/down regulated.

Figure 4

GO analysis of DE miR target genes. (A) Biological processes for differential miRNAs. (B) cellular components for differential miRNAs. (C) Molecular functions for differential miRNAs.

Figure 5

miRNA-mRNA-miRNA interaction network of differentially expressed miRNAs using MirNet online tool.