miR-215 Targeting Novel GenesEREG,NIPAL1 andPTPRURegulates the Resistance toE.coliF18 in Piglets

Previous research has revealed that miR-215 might be an important miRNA regulating weaned piglets' resistance toEscherichia 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 andE. coliinfection was explored by RNAi technology, combined withE. colistimulation 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 targetEREG,NIPAL1andPTPRUgenes. 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 afterE. coliF18 stimulation, while cytokines levels of TNF-alpha and IL-1 beta 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 toE. coliF18 by targetingEREG,NIPAL1andPTPRUgenes. This study is the first to annotate new biological functions ofEREG,NIPAL1andPTPRUgenes in pigs, and provides a new experimental basis and reference for the research of piglets disease-resistance breeding.