Integrated Analysis of Transcriptome Profiles and lncRNA-miRNA-mRNA Competing Endogenous RNA Regulatory Network to Identify Biological Functional Effects of Genes and Pathways Associated with Johne's Disease in Dairy Cattle

Abstract

Paratuberculosis or Johne's disease (JD), a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes huge economic losses and reduces animal welfare in dairy cattle herds worldwide. At present, molecular mechanisms and biological functions involved in immune responses to MAP infection of dairy cattle are not clearly understood. Our purpose was to integrate transcriptomic profiles and competing endogenous RNA (ceRNA) network analyses to identify key messenger RNAs (mRNAs) and regulatory RNAs involved in molecular regulation of peripheral blood mononuclear cells (PBMCs) for MAP infection in dairy cattle. In total, 28 lncRNAs, 42 miRNAs, and 370 mRNAs were identified by integrating gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In this regard, we identified 21 hub genes (CCL20, CCL5, CD40, CSF2, CXCL8, EIF2AK2, FOS, IL10, IL17A, IL1A, IL1B, IRF1, MX2, NFKB1, NFKBIA, PTGS2, SOCS3, TLR4, TNF, TNFAIP3, and VCAM1) involved in MAP infection. Furthermore, eight candidate subnets with eight lncRNAs, 29 miRNAs, and 237 mRNAs were detected through clustering analyses, whereas GO enrichment analysis of identified RNAs revealed 510, 22, and 11 significantly enriched GO terms related to MAP infection in biological process, molecular function, and cellular component categories, respectively. The main metabolic-signaling pathways related to MAP infection that were enriched included the immune system process, defense response, response to cytokine, leukocyte migration, regulation of T cell activation, defense response to bacterium, NOD-like receptor, B cell receptor, TNF, NF-kappa B, IL-17, and T cell receptor signaling pathways. Contributions of transcriptome profiles from MAP-positive and MAP-negative sample groups plus a ceRNA regulatory network underlying phenotypic differences in the intensity of pathogenicity of JD provided novel insights into molecular mechanisms associated with immune system responses to MAP infection in dairy cattle.

Keywords: MAP infection; ceRNA regulatory network; dairy cattle; immune responses; paratuberculosis; transcriptome analysis.

Figure 1

Venn diagram of common significant genes for (A) RNA-Seq and (B) microarray datasets related to MAP infection in dairy cattle.

Figure 2

Top significant mRNA/gene ontology (GO) terms including biological process (A), molecular function (B), and cellular component (C) enriched using mRNAs/genes associated with MAP infection in dairy cattle. The numbers indicate the number of genes associated with each pathway.

Figure 3

Top KEGG pathways enriched using significant mRNAs/genes associated with MAP infection in dairy cattle.

Figure 4

The main lncRNA–miRNA–mRNA ceRNA regulatory network and its subnets. Octagonal nodes represent lncRNAs, triangle nodes represent miRNAs, and quadrilateral nodes represent mRNAs/genes. Edges indicate interactions between nodes.

Figure 5

Schematic of the workflow used to construct the lncRNA–miRNA–mRNA ceRNA regulatory network involved in MAP infection. Regulatory RNAs were obtained from microarray and RNA-Seq data analyses, plus literature mining. Protein–protein interaction network (PPI), gene regulatory network (GRN), and ceRNA regulatory network were constructed using the STRING biological database and Cytoscape software.