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

Abstract

22In this study, we present a comprehensive analysis of the hepatic miRNA transcriptome in 23 mice suffering from experimental primary alveolar echinococcosis (AE), a parasitic infection 24 caused upon ingestion of Echinococcus multilocularis (E. multilocularis) eggs. At one month 25 post-infection, infected C57BL/6 mice, along with non-infected control mice, were 26 euthanized. Subsequently livers were collected and used for small RNA library preparation 27 and next-generation sequencing (NGS). The most significantly dysregulated hepatic miRNAs 28 were validated by Stem-loop RT-qPCR. We identified 28 miRNAs with significantly altered 29 expression levels upon infection with E. multilocularis. Of these, 9 were up-regulated (fold 30 change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected 31 controls. In infected liver tissues, mmu-miR-148a-3p and mmu-miR-101b-3p were 8-and 6-32 fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 33 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels 34 were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 35 1.8). Down-regulated miRNAs were highly enriched in Reactome and KEGG pathways of 36 angiogenesis and fatty acids biosynthesis. Moreover, relative mRNA expression levels of 37 three pro-angiogenic (VEGFA, MTOR and HIF1-α) and two lipogenic (FASN and ACSL1) 38 genes were significantly higher in livers of E. multilocularis infected mice. 39Lastly, we studied the issue related to functionally mature arm selection preference (5p and/or 40 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm 41 selection preferences in normal versus infected liver tissues. Our study provides first evidence 42 of miRNA involvement in liver pathogenesis during AE. Our future research will focus on the 43 characterization of miRNA transcriptome patterns in more advanced AE-stages towards the 44 assessment of microRNA therapy for AE, and experimentally address functional 45 characteristics of selected features presently found. 48of microRNAs (miRNAs), a class of small non-coding RNAs involved in negative regulation 49 of gene expression. Herein, we revealed that significant alterations of miRNA expression 50 occurred in murine liver subsequently to experimental infection with Echinococcus 51 multilocularis (E. multilocularis) eggs when compared to non-infected controls. 52At the early stage of murine AE, hepatic miRNAs were mainly downregulated. Respective 53 target genes of the most extensively downregulated miRNAs were involved in angiogenesis 54 and fatty acid synthesis. Indeed, angiogenic and lipogenic genes were found to be 55 significantly higher expressed in E. multilocularis infected livers relative to non-infected 56 controls. These boosted cellular pathways are advantageous for development of the E. 57 multilocularis metacestodes, since this larval stage is not able to undertake de novo fatty acid 58 synthesis, and angiogenesis ...