Analysing the promoters and miRNAs of key upregulated genes in resistant chicken lines co-infected with Eimeria maxima/Clostridium perfringens: an in silico approach

Abstract

Necrotic enteritis (NE) is one of the most economically important diseases affecting the poultry industry worldwide. Understanding the regulatory mechanisms that control host resistance to NE is essential for identifying molecular markers associated with disease resilience. Promoter regions and microRNAs (miRNAs) play critical roles in regulating gene expression and coordinating immune responses; therefore, their characterization may provide valuable insights into the molecular basis of disease resistance. The present study aimed to identify key regulatory elements, including promoter motifs, transcription factors, and miRNAs, associated with upregulated genes in resistant White Leghorn chicken line co-infected with Eimeria maxima and Clostridium perfringens . In this study, gene networks, gene ontologies, biological pathways, identification of hub genes, promoter interactions and interactions between miRNAs and hub genes in the spleens of Marek’s disease-resistant Leghorn chickens infected with C. perfringens and E. maxima were investigated. Bioinformatics tools were used to conduct this study. For the gene interaction analysis, 216 genes with a p -value of less than 0.01 and a fold change of greater than two relative to the control group were used. miRNA targets of hub genes were predicted using miRDB, and only high-confidence interactions (score > 80) were considered. The results revealed that a substantial proportion of the analyzed upregulated genes were involved in immune-related processes, and several hub genes, including CD molecules and cytokines, were identified. In addition, network analysis revealed hub genes and their interactions led to the identification of three functional clusters. The KEGG pathways of cell adhesion molecules, the intestinal immune network for IgA production, and cytokine-cytokine receptor interactions were commonly identified across all three clusters, indicating their importance in the network. Promoter motif analysis revealed notable binding sites for the upregulated hub genes. Six effective motif binding sites were identified for the upregulated genes. The transcription factors MIX-A, PHOX2B, ZNF384, KLF15, ZNF610, and PATZ1 were found at all six of these loci. The top five gene ontology terms predicted for these motifs were protein transport, translation, regulation of transcription, and DNA dependent, which are common biological processes. The integrated analysis of upregulated genes in resistant chickens revealed that immune-related pathways, key hub genes, transcription factor binding motifs, and miRNA-gene interactions collectively contribute to the host response against necrotic enteritis. The identified regulatory network suggests that both transcriptional and post-transcriptional mechanisms are involved in disease resistance. In particular, hub genes such as TNFSF13B and PTPRC emerged as central regulatory components and may represent promising candidates for future functional validation and biomarker development. These findings provide insights into the molecular mechanisms underlying the immune response to necrotic enteritis in poultry, which could contribute to the development of new strategies for disease control.