The regulation roles of miR-125b, miR-221 and miR-27b in porcine Salmonella infection signalling pathway.

Min Yao,Jingbo Luo,Jun Yang,Tinghua Huang,Weihua Gao,Xiongyan Liang

doi:10.1042/bsr20160243

Abstract

miRNAs are non-coding RNA molecules typically 18–22 nucleotides long that can suppress the expression of their target genes. Several laboratories have attempted to identify miRNAs from the pig that are involved in Salmonella infection. These bioinformatics strategies using the newly available genomic sequence are generally successful. Here, we report an in silico identification of miRNAs in pig focusing on the Salmonella infection pathway, and further investigated the differential expression of those miRNAs by quantitative real-time PCR during pre- and post-natal stage of Salmonella inoculation from the peripheral blood of commercially breed pigs. We identified 29 miRNAs that have predicted targets in the Salmonella infection pathway and nine of them were not yet described in pig. In addition, the expression of nine selected miRNAs was validated in the peripheral blood by northern blotting. Through expression analyses, differences were found between pre- and post-natal stages of Salmonella inoculation for miR-221, miR-125b and miR-27b—all of them were suppressed 2 days after Salmonella inoculation. The predicted targets of those three miRNAs were validated by luciferase reporter assays. We show that FOS is a direct target of miR-221, miR-125b can suppress MAPK14, and miR-27b can target IFNG. These findings will be helpful in understanding the function and processing of these miRNAs in Salmonella infection. The miRNA differentially expressed in the peripheral blood of commercial breed pigs suggest that it can be used as genetic markers for salmonella infection resistance in pigs.

Highlights

Salmonella colonization of pigs can lead to enterocolitis, and the bacteria often establish a carrier status in the host [1]
MiRNAs predict target genes involved in the Salmonella infection pathway Porcine miRNA targets that were conserved across multiple organisms were identified with TargetScan searches for segments of 2–8 bases from the 5’ end of the miRNA using 3’ UTR alignments, which is referred to as the ‘miRNA seed’
We identified miRNAs and target genes that mediate the effect of Salmonella infection using the following criteria: (1) the miRNA was predicted target genes involved in the Salmonella infection pathway; (2) the miRNA is expressed in either peripheral blood or lymph nodes; (3) the miRNA is differentially expressed in the Salmonella inoculation experiment in peripheral blood samples and (4) the miRNA regulation of its target gene can be validated by the luciferase reporter assay

Summary

Introduction

Salmonella colonization of pigs can lead to enterocolitis, and the bacteria often establish a carrier status in the host [1]. Salmonella colonization is subclinical but can decrease the quality of pork, and has a negative economic impaction on the meatpacking industry. Reducing the incidence and severity of salmonellosis requires identification of the immune genes and pathways responsible for enhanced disease resistance and pathogen clearance [4]. In addition to protein-coding genes, miRNAs play important roles in the regulation of Salmonella infection. The miRNAs are one of the most important families of noncoding RNAs (ncRNAs), and they have important sequence-specific post transcriptional regulators of gene expression [5]. The mammalian inflammatory response to Salmonella infection involves the induction of several miRNAs. The mammalian inflammatory response to Salmonella infection involves the induction of several miRNAs This process must be carefully regulated to achieve pathogen clearance and prevent the consequences of regulated expression

Methods

Results

Discussion

Conclusion