Abstract:

Objective To analyze the microRNA (miRNA) expression profiles in the liver of mice infected with Capillaria hepatica, screening deferentially expressed miRNA, to provide basis for research on the mechanism of liver fibrosis caused by C. hepatica infection. Methods Six male BALB/c mice were randomly assigned to the C. hepatica infection group and control group, with 3 mice in each group. Each of the infection group mice was infected with 20 infective eggs of C. hepatica by gavage. For the control group, mice were given the same volume of saline. At 35 days post-infection, mice liver tissues from the two groups were collected for histo-section and hematoxylin-eosin（HE） staining to observe the pathological changes microscopically. Mice liver tissue total RNA was extracted and purified for construction of liberary and high throughput sequencing. Bioinformatics analysis was used to screen differentially expressed miRNA, estimate target genes and perform gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment. Real-time quantitative PCR (qRT-PCR) was used to validate part of differentially expressed miRNAs. Results The liver tissue sections stained with HE showed egg-granulomatous lesion in the livers of infection group, accompanying with significant inflammatory cell infiltration, while the hepatocytes in control group remained morphologically intact, with no inflammatory cell infiltration, degeneration, necrosis and fibrosis. Based on miRNA sequencing, the screening found 16 differentially expressed miRNAs, among them 4 were expression up-regulated with elevating over 2-fold, of which mmu-miR-129-5p expression was elevated 4-fold; 12 miRNAs expression were down-regulated with lowering more than 50%, of which mmu-miR-21a-3p expression was lowered by more than 10%. The GO analysis of the miRNA target genes showed that the enriched genes were mainly involved in signal transduction, protein binding, transferase activity, G protein coupled receptor signaling pathway, negative regulation of apoptotic process, positive regulation of cell proliferation and regulation of transcription. The KEGG pathway analysis showed that the enriched pathways were Hippo signaling pathway, Ras signaling pathway, mTOR signaling pathway, sphingolipid signaling pathway and Wnt signaling pathway. qRT-PCR assay indicated that the relative expression level of up-regulated genes, mmu-miR-21a-3p and mmu-miR-181a-1-3p, in the infection group were 0.70 ± 0.30 and 0.68 ± 0.27, while the relative expression level of down-regulated genes, mmu-miR-409-5p and mmu-miR-129-5p, were 1.27 ± 0.24 and 2.61 ± 0.52, respectively, which were consistent with the changing trend expected by sequencing in comparison with the reference genes. Conclusion C. hepatica infection caused changes in miRNA expression profile in the liver tissue of infected mice. It was demonstrated mmu-miR-409-5p and mmu-miR-129-5p may be the potential genes involving in liver fibrosis.

Key words: Capillaria hepatica, miRNA, Differential expression, Liver fibrosis