Effects of mutations in porcine miRNA-215 precursor sequences on miRNA-215 regulatory function

Abstract

MicroRNAs (miRNAs) play an important role in animal growth and disease development, and sequence variation in microRNAs can alter their functions. Herein, we explored the effects of mutations in the miRNA-215 precursor sequence on the miRNA-215 regulatory network and resistance to Escherichia coli (E. coli). Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to detect sequence variations in Sutai and Meishan pigs. The miR-192 precursor sequence was not mutated, but the miR-215 precursor included an AT insertion mutation at position 6 (start from the first base of the miR-215 precursor) and a C/T mutation at position 43. Wild-type (WT) and mutant miR-215 precursor expression vectors were constructed to investigate the effects of sequence variation on expression of miR-215 and its target genes DLG5 and ALCAM, cytokine levels and E. coli adhesion. Compared with the WT control group, cells harbouring the C/T mutant vector displayed reduced miR-215 expression, increased target gene expression, elevated cytokine levels and rising E. coli adhesion, whereas cells harbouring the AT insertion mutant vector were not significantly changed. The sequence variation in the miRNA-215 precursor may affect the miRNA-215 regulatory network, and alter the stability of intestinal epithelial cells (IPEC-J2 cells) and resistance to E. coli. Our findings provide guidance for future research on the regulatory mechanisms of miR-215 in porcine resistance to E. coli F18, and identifying effective genetic markers against this organism.