Differential microRNA expression profile of Trichinella spiralis larvae after exposure to the host small intestinal milieu

Abstract

The process by which Trichinella spiralis muscle larvae are activated to infect the intestine after exposure to the host small intestinal milieu is crucial for the successful establishment of T. spiralis infection. However, the molecular mechanism underlying the invasion of intestinal epithelial cells by T. spiralis has not been elucidated. MicroRNAs are a class of small noncoding RNAs that participate in parasite growth and development, pathogenic processes, and host-parasite interactions. In the present study, the differential expression profile of miRNAs in T. spiralis after exposure to the mouse small intestinal milieu was analysed using Solexa high-throughput sequencing technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to analyse the functions of miRNA target genes, and dual-luciferase reporter assays were subsequently applied to test the regulatory effects of one significantly decreased miRNA (let-7) on its four predicted target genes. In total, 2,000 known miRNAs (930 upregulated and 1070 downregulated) and 43 novel miRNAs (22 upregulated and 21 downregulated) were found to be differentially expressed in intestinal larvae, compared with muscle larvae. The KEGG pathway analysis showed that the predicted target genes of differentially expressed miRNAs were involved in 299 different pathways, and the top 10 pathways were metabolic pathways, biosynthesis of secondary metabolites, neuroactive ligand-receptor interaction, lysosome, focal adhesion, purine metabolism, starch and sucrose metabolism, tight junction, carbohydrate digestion and absorption, and pathways in cancer. As one of the most widely studied miRNA families, the expression of let-7 was significantly decreased in T. spiralis after exposure to host small intestinal milieu. A dual-luciferase reporter assay revealed that neuropeptide Y receptor type 6 and carboxypeptidase E were direct target genes of let-7, and were downregulated by binding with their 3′ UTR. GO function analysis showed that carboxypeptidase E had multiple enzymatic activities, suggesting that it might participate in cell membrane damage and larval invasion. These data suggest that the differentially expressed miRNAs in T. spiralis might have a regulatory role in the invasion of host intestinal epithelial cells. This study provides a new insight into the molecular mechanisms of invasion by T. spiralis and the regulatory functions of miRNAs in host-Trichinella interactions.