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.
Keywords: E. coli resistance; RNA-seq; miR-215; pig; promoter region; target genes.