Regulatory network of miRNA, lncRNA, transcription factor and target immune response genes in bovine mastitis

Abstract

Pre- and post-transcriptional modifications of gene expression are emerging as foci of disease studies, with some studies revealing the importance of non-coding transcripts, like long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). We hypothesize that transcription factors (TFs), lncRNAs and miRNAs modulate immune response in bovine mastitis and could potentially serve as disease biomarkers and/or drug targets. With computational analyses, we identified candidate genes potentially regulated by miRNAs and lncRNAs base pair complementation and thermodynamic stability of binding regions. Remarkably, we found six miRNAs, two being bta-miR-223 and bta-miR-24-3p, to bind to several targets. LncRNAs NONBTAT027932.1 and XR_003029725.1, were identified to target several genes. Functional and pathway analyses revealed lipopolysaccharide-mediated signaling pathway, regulation of chemokine (C-X-C motif) ligand 2 production and regulation of IL-23 production among others. The overarching interactome deserves further in vitro/in vivo explication for specific molecular regulatory mechanisms during bovine mastitis immune response and could lay the foundation for development of disease markers and therapeutic intervention.

Figure 1

Step-by-step pipeline of analysis procedures (a) and number of articles mentioning each of the 16 target bovine mastitis genes (b). Figure created with Microsoft PowerPoint (2013) (https://www.microsoft.com/en-us/microsoft-365/powerpoint).

Figure 2

Network of 16 target bovine mastitis genes created using STRING (string-db.org). The Ensembl ID in the network refers to IL-6. Edge color legend (blue: from curated database, pink: experimentally determined, green: text mining, brown: co-expression).

Figure 3

Venn Diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/) for 14 out of 16 target bovine mastitis genes showing the number of miRNA binding to the 3′ UTR, CDS, and 5′ UTR. The overlapping region of each diagram represents miRNA that bind to all three regions of the target gene.

Figure 4

miRNA predicted to bind to three or more target genes. The red bars are miRNA predicted to bind to 3′ UTR, CDS, and 5′ UTR. The blue bars represent miRNA binding to two of the three regions. The green bars are miRNA predicted to bind to one of the three regions. Figure created with Microsoft Excel (2013) (https://www.microsoft.com/en-us/microsoft-365/excel).

Figure 5

The 20 lncRNA and the number of target genes they are predicted to bind to. The darkest shade of blue corresponds to the lowest (-) ndG and the lightest shade of blue corresponds to the highest (-) ndG. Figure created with Microsoft Excel (2013) (https://www.microsoft.com/en-us/microsoft-365/excel).

Figure 6

The biological processes and the number of target genes involved (a); and pathways and corresponding number of target genes (b). In both (a) and (b), black bars indicate biological processes/pathways predicted with the lowest p-value; lightest red bar represents predictions with the greatest p-value. Figure created with Microsoft Excel (2013) (https://www.microsoft.com/en-us/microsoft-365/excel).

Figure 7

miRNA-lncRNA predicted binding with their normalized binding free energy (ndG). The black bars indicate the entire miRNA binds to the lncRNA; the lighter the green bar, the less complementary basing between miRNA and lncRNA. Figure created with Microsoft Excel (2013).

Figure 8

Network interactome of miRNA, their target genes, and the gene ontologies of the target genes. The pink diamonds are miRNA binding to a single target gene while the red diamonds are miRNA binding to two or more target genes. The green circles are the 16 target genes and the blue rectangles are the biological processes, molecular functions, cellular components, and pathways. (Cytoscape version 3.7.2; https://cytoscape.org/).

Figure 9

A network incorporating lncRNA, their target genes and corresponding gene ontologies. The green triangles are the lncRNA, red circles represent the target bovine mastitis genes, and the pink rectangles are the biological processes, molecular functions, cellular components, and pathways (Cytoscape version 3.7.2; https://cytoscape.org/).

Figure 10

Network interactome of transcription factors (TFs), target genes, and gene ontologies. The red triangles are TFs; green circles are gene targets; while the ovals represent the biological processes, molecular functions, cellular components, and pathways (Cytoscape version 3.7.2; https://cytoscape.org/).

Figure 11

A network incorporating miRNA, lncRNA, TFs, target genes, and gene ontologies. The red arrowheads represent miRNA; purple diamonds represent lncRNA; teal triangles represent TFs; green circles represent target genes, and the blue rectangles biological processes, molecular functions, cellular components, and pathways (Cytoscape version 3.7.2; https://cytoscape.org/).