1- Animal science research institute of Iran (ASRI), Agricultural Research, Education, and Extension Organization (AREEO)
2- University of Jiroft
2- University of Jiroft
Abstract: (45 Views)
Extended abstract
Introduction
Bovine mastitis is one of the most common and costly infectious diseases affecting the global dairy industry and is responsible for considerable economic losses each year through reduced milk yield, compromised milk quality, increased treatment costs, premature culling, and extensive antibiotic use. The disease is characterized by pronounced inflammatory responses in the mammary gland, resulting from complex interactions between invading pathogens, the host innate and adaptive immune systems, and genetic and epigenetic regulatory mechanisms. Among mastitis‑associated pathogens, Escherichia coli is a major cause of acute mastitis and induces a rapid inflammatory response that leads to tissue damage and marked alterations in gene expression patterns.
High‑throughput omics technologies, particularly microarray and RNA‑seq approaches, have demonstrated that mastitis is accompanied by extensive transcriptional reprogramming involving genes related to innate immunity, inflammation, energy metabolism, cell cycle control, and tissue remodeling. However, most previous studies have focused primarily on lists of differentially expressed genes, while the underlying gene interaction networks and the contribution of microRNAs (miRNAs) as post‑transcriptional regulators have received less attention. miRNAs are capable of simultaneously regulating multiple mRNA targets and, as a result, can modulate key immune and metabolic pathways that influence the intensity and progression of inflammatory responses. Therefore, the present study aimed to identify central hub genes and regulatory miRNAs involved in E. coli‑induced mastitis in dairy cows and to explore their roles within immune‑ and metabolism‑related molecular networks using an integrated bioinformatics framework.
Materials and Methods
Gene expression data from bovine mammary glands affected by Escherichia coli‑induced mastitis were obtained from the Gene Expression Omnibus (GEO) database (accession number GSE15020). Differentially expressed genes were identified using GEO2R, applying a threshold of p < 0.05 and |LogFC| > 1 to ensure statistical significance. Protein–protein interaction (PPI) analysis was performed using the STRING database, and the resulting network was visualized and analyzed in Cytoscape. Hub genes were identified using the cytoHubba plugin based on multiple topological algorithms, including Degree, MCC, MNC, and DMNC, to increase confidence in hub selection. Subsequently, miRNAs targeting these hub genes were predicted using the miRWalk database, and miRNAs shared among multiple hub genes were retained for further analysis. This strategy enabled the construction of integrated gene–miRNA interaction networks and facilitated biological interpretation of the regulatory mechanisms involved in bovine mastitis.
Results
A total of 1,023 genes were identified as differentially expressed, including 612 upregulated and 411 downregulated genes following E. coli infection. PPI network analysis highlighted ten genes GAPDH, ALB, CXCL8, IL1B, CCL2, EGFR, PTPRC, PARP12, IFITM3, and XAF1as central hubs within the network. These genes are mainly associated with inflammatory signaling, chemokine‑mediated immune responses, regulation of cell survival, and immune cell activation.
Among these hubs, IL1B represents a central mediator of fever and inflammation and is widely recognized as a major pro‑inflammatory cytokine involved in fever induction, increased vascular permeability, and activation of immune cells. CXCL8 plays a key role in neutrophil recruitment and activation and contributes substantially to the early immune response against bacterial infection. In addition, ALB, identified as a hub gene in the network, may have potential value as a milk‑based biomarker through its association with positive acute‑phase proteins during mastitis.
miRNA analysis identified 45 miRNAs that commonly targeted multiple hub genes. Notably, bta‑miR‑21‑3p targets EGFR and IL1B and is involved in regulation of the PI3K/AKT/NF‑κB signaling pathway, suggesting a role in amplifying inflammatory responses. bta‑miR‑145 may contribute to balancing inflammation and tissue repair by regulating IL1B expression and inhibiting proliferation of mammary epithelial (Mac‑T) cells. Furthermore, bta‑miR‑138 appears to modulate antiviral and inflammatory responses through inhibition of the p65/NF‑κB pathway and regulation of PARP12 and EGFR. Overall, these results indicate that miRNAs act as coordinated regulators of inflammatory and metabolic pathways during mastitis.
Conclusion
Bovine mastitis is a complex, multifactorial condition driven by dynamic interactions among genes, miRNAs, and interconnected molecular pathways. By integrating differential expression analysis with network‑based and miRNA regulatory analyses, this study identified key molecular regulators involved in E. coli‑induced mastitis. The findings highlight the importance of immune‑related pathways, including JAK/STAT, interferon signaling, and NF‑κB, as well as EGFR‑associated metabolic and mitochondrial processes. These results provide a useful foundation for the development of molecular biomarkers, diagnostic tools, and breeding strategies aimed at improving mastitis resistance in dairy cattle.
Introduction
Bovine mastitis is one of the most common and costly infectious diseases affecting the global dairy industry and is responsible for considerable economic losses each year through reduced milk yield, compromised milk quality, increased treatment costs, premature culling, and extensive antibiotic use. The disease is characterized by pronounced inflammatory responses in the mammary gland, resulting from complex interactions between invading pathogens, the host innate and adaptive immune systems, and genetic and epigenetic regulatory mechanisms. Among mastitis‑associated pathogens, Escherichia coli is a major cause of acute mastitis and induces a rapid inflammatory response that leads to tissue damage and marked alterations in gene expression patterns.
High‑throughput omics technologies, particularly microarray and RNA‑seq approaches, have demonstrated that mastitis is accompanied by extensive transcriptional reprogramming involving genes related to innate immunity, inflammation, energy metabolism, cell cycle control, and tissue remodeling. However, most previous studies have focused primarily on lists of differentially expressed genes, while the underlying gene interaction networks and the contribution of microRNAs (miRNAs) as post‑transcriptional regulators have received less attention. miRNAs are capable of simultaneously regulating multiple mRNA targets and, as a result, can modulate key immune and metabolic pathways that influence the intensity and progression of inflammatory responses. Therefore, the present study aimed to identify central hub genes and regulatory miRNAs involved in E. coli‑induced mastitis in dairy cows and to explore their roles within immune‑ and metabolism‑related molecular networks using an integrated bioinformatics framework.
Materials and Methods
Gene expression data from bovine mammary glands affected by Escherichia coli‑induced mastitis were obtained from the Gene Expression Omnibus (GEO) database (accession number GSE15020). Differentially expressed genes were identified using GEO2R, applying a threshold of p < 0.05 and |LogFC| > 1 to ensure statistical significance. Protein–protein interaction (PPI) analysis was performed using the STRING database, and the resulting network was visualized and analyzed in Cytoscape. Hub genes were identified using the cytoHubba plugin based on multiple topological algorithms, including Degree, MCC, MNC, and DMNC, to increase confidence in hub selection. Subsequently, miRNAs targeting these hub genes were predicted using the miRWalk database, and miRNAs shared among multiple hub genes were retained for further analysis. This strategy enabled the construction of integrated gene–miRNA interaction networks and facilitated biological interpretation of the regulatory mechanisms involved in bovine mastitis.
Results
A total of 1,023 genes were identified as differentially expressed, including 612 upregulated and 411 downregulated genes following E. coli infection. PPI network analysis highlighted ten genes GAPDH, ALB, CXCL8, IL1B, CCL2, EGFR, PTPRC, PARP12, IFITM3, and XAF1as central hubs within the network. These genes are mainly associated with inflammatory signaling, chemokine‑mediated immune responses, regulation of cell survival, and immune cell activation.
Among these hubs, IL1B represents a central mediator of fever and inflammation and is widely recognized as a major pro‑inflammatory cytokine involved in fever induction, increased vascular permeability, and activation of immune cells. CXCL8 plays a key role in neutrophil recruitment and activation and contributes substantially to the early immune response against bacterial infection. In addition, ALB, identified as a hub gene in the network, may have potential value as a milk‑based biomarker through its association with positive acute‑phase proteins during mastitis.
miRNA analysis identified 45 miRNAs that commonly targeted multiple hub genes. Notably, bta‑miR‑21‑3p targets EGFR and IL1B and is involved in regulation of the PI3K/AKT/NF‑κB signaling pathway, suggesting a role in amplifying inflammatory responses. bta‑miR‑145 may contribute to balancing inflammation and tissue repair by regulating IL1B expression and inhibiting proliferation of mammary epithelial (Mac‑T) cells. Furthermore, bta‑miR‑138 appears to modulate antiviral and inflammatory responses through inhibition of the p65/NF‑κB pathway and regulation of PARP12 and EGFR. Overall, these results indicate that miRNAs act as coordinated regulators of inflammatory and metabolic pathways during mastitis.
Conclusion
Bovine mastitis is a complex, multifactorial condition driven by dynamic interactions among genes, miRNAs, and interconnected molecular pathways. By integrating differential expression analysis with network‑based and miRNA regulatory analyses, this study identified key molecular regulators involved in E. coli‑induced mastitis. The findings highlight the importance of immune‑related pathways, including JAK/STAT, interferon signaling, and NF‑κB, as well as EGFR‑associated metabolic and mitochondrial processes. These results provide a useful foundation for the development of molecular biomarkers, diagnostic tools, and breeding strategies aimed at improving mastitis resistance in dairy cattle.
Type of Study: Research |
Subject:
ژنتیک و اصلاح نژاد دام
Received: 2025/11/22 | Accepted: 2026/05/4
Received: 2025/11/22 | Accepted: 2026/05/4
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