Abstract
Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets’ resistance to Escherichia coli (E. coli) F18. In this study, target genes of miR-215 were identified by RNA-seq, bioinformatics analysis and dual luciferase detection. The relationship between target genes and E. coli infection was explored by RNAi technology, combined with E. coli stimulation and enzyme linked immunosorbent assay (ELISA) detection. Molecular regulating mechanisms of target genes expression were analyzed by methylation detection of promoter regions and dual luciferase activity assay of single nucleotide polymorphisms (SNPs) in core promoter regions. The results showed that miR-215 could target EREG, NIPAL1 and PTPRU genes. Expression levels of three genes in porcine intestinal epithelial cells (IPEC-J2) in the RNAi group were significantly lower than those in the negative control pGMLV vector (pGMLV-NC) group after E. coli F18 stimulation, while cytokines levels of TNF-α and IL-1β in the RNAi group were significantly higher than in the pGMLV-NC group. Variant sites in the promoter region of three genes could affect their promoter activities. These results suggested that miR-215 could regulate weaned piglets’ resistance to E. coli F18 by targeting EREG, NIPAL1 and PTPRU genes. This study is the first to annotate new biological functions of EREG, NIPAL1 and PTPRU genes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.
Highlights
MicroRNAs are a class of conserved endogenous noncoding small RNAs with about22 nt length
By detecting the transcription level of miR-215 in IPEC-J2 cells, the mRNA expression level of the miR-215 inhibitor group had a 0.2-fold change compared to that of the negative control (NC) group (p < 0.01) (Figure 2C). These results indicated that the IPEC-J2 cell line with miR-215 stable interference was successfully constructed and could be used for further studies
We first verified the relationship between the expression of miR-215 and E. coli stimulation in IPEC-J2 cells and found that the expression level of miR-215 was significantly stimulation in IPEC-J2 cells and found that the expression level of miR-215 was significantly downdown-regulated in E. coli F18ac-stimulated IPEC-J2 cells, which suggested that miR-215 may play an regulated in IPEC-J2 cells, which suggested that miR-215 may play an important regulatory role in the process of E. coli infection. miRNAs indirectly affect phenotype and important the process of E. coli infection
Summary
They are widely distributed in cells of plants, animals, nematodes and humans and play important roles in biological processes by regulating gene expression at the post-transcriptional level [1]. It has been found that miRNAs are widely involved in various organs’ development, cell proliferation and differentiation, migration and apoptosis, lipogenesis metabolism, insulin secretion and other physiological activities [2,3,4,5]. They play important regulatory roles in pathological processes such as tumor, viral and bacterial diseases [6,7,8,9]. Recent research identified molecular networks within genes, and miRNAs were candidate factors determining adipogenesis, fatness, and selected fattening characteristics in pigs [10].
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