Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro

PERÉZ, M.; SPILLIOTIS, M.; REGO, N.; MACCHIAROLI, N.; KAMENETZKY, L.; HOLROYD N.; CUCHER, M.; BREHM, K.; ROSENZVIT, M.

PLOS NEGLECTED TROPICAL DISEASES

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2019

1935-2735

Echinococcosis represents a major public health problem worldwide and is considered31 a neglected disease by the World Health Organization. The etiological agents are32 Echinococcus tapeworms, which display elaborate developmental traits that imply a33 complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory34 RNAs, are involved in the regulation of many biological processes such as35 development and metabolism. They act through the repression of messenger RNAs36 (mRNAs) usually by binding to the 3? untranslated region (3?UTR). Previously, we37 described the miRNome of several Echinococcus species and found that miRNAs are38 highly expressed in all life cycle stages, suggesting an important role in gene39 expression regulation. However, studying the role of miRNAs in helminth biology40 remains a challenge. To develop methodology for functional analysis of miRNAs in41 tapeworms, we performed miRNA knockdown experiments in primary cell cultures of42 Echinococcus multilocularis, which mimic the development of metacestode vesicles43 from parasite stem cells in vitro. First, we analysed the miRNA repertoire of E.44 multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian45 miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs.Using genomic information and bioinformatic algorithms for 46 miRNA binding prediction,47 we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of48 miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that49 block miRNA function. To this end, we evaluated a variety of chemically modified anti50miRs for miR-71 knockdown. Electroporation of primary cells with 2?-O-methyl modified51 anti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analyses showed52 that several predicted miR-71 targets were up-regulated in anti-miR-treated primary53 cells, including genes potentially involved in parasite development, host parasite54 interaction, and several genes of as yet unknown function. Notably, miR-71-silenced55 primary cell cultures showed a strikingly different phenotype as from control cells and56 did not develop into fully mature metacestodes. These findings indicate an important57 function of miR-71 in Echinococcus development and provide, for the first time,58 methodology to functionally study miRNAs in a tapeworm.