Regulation of Hepatic MicroRNAs in Response to Early Stage Echinococcus multilocularis Egg Infection in C57BL/6 mice

Abstract

In this study, we present a comprehensive analysis of the hepatic miRNA transcriptome in mice suffering from experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of Echinococcus multilocularis (E. multilocularis) eggs. At one month post-infection, infected C57BL/6 mice, along with non-infected control mice, were euthanized. Subsequently livers were collected and used for small RNA library preparation and next-generation sequencing (NGS). The most significantly dysregulated hepatic miRNAs were validated by Stem-loop RT-qPCR. We identified 28 miRNAs with significantly altered expression levels upon infection with E. multilocularis. Of these, 9 were up-regulated (fold change (FC) [≥] 1.5) and 19 were down-regulated (FC [≤] 0.66) as compared to the non-infected controls. In infected liver tissues, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). Down-regulated miRNAs were highly enriched in Reactome and KEGG pathways of angiogenesis and fatty acids biosynthesis. Moreover, relative mRNA expression levels of three pro-angiogenic (VEGFA, MTOR and HIF1-) and two lipogenic (FASN and ACSL1) genes were significantly higher in livers of E. multilocularis infected mice. Lastly, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. Our study provides first evidence of miRNA involvement in liver pathogenesis during AE. Our future research will focus on the characterization of miRNA transcriptome patterns in more advanced AE-stages towards the assessment of microRNA therapy for AE, and experimentally address functional characteristics of selected features presently found.\n\nAuthor SummaryVarious infectious diseases in humans have been associated with altered expression patterns of microRNAs (miRNAs), a class of small non-coding RNAs involved in negative regulation of gene expression. Herein, we revealed that significant alterations of miRNA expression occurred in murine liver subsequently to experimental infection with Echinococcus multilocularis (E. multilocularis) eggs when compared to non-infected controls. At the early stage of murine AE, hepatic miRNAs were mainly downregulated. Respective target genes of the most extensively downregulated miRNAs were involved in angiogenesis and fatty acid synthesis. Indeed, angiogenic and lipogenic genes were found to be significantly higher expressed in E. multilocularis infected livers relative to non-infected controls. These boosted cellular pathways are advantageous for development of the E. multilocularis metacestodes, since this larval stage is not able to undertake de novo fatty acid synthesis, and angiogenesis allows the larvae to be periparasitically supplied by oxygen and nutrients and to get rid of waste products. More research on the miRNA transcriptome at more advanced infection-stages, and on the role of angiogenesis in E. multilocularis larval growth and metastasis, is required to assess the usefulness of microRNA- and anti-angiogenic therapies against E. multilocularis infection.