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

PÉREZ MG; SPILIOTIS M; REGO N; MACCHIAROLI N; KAMENETZKY L; HOLROYD N; CUCHER M; BREHM K; ROSENZVIT M C

PLOS NEGLECTED TROPICAL DISEASES

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2019

1935-2735

Echinococcosis represents a major public health problem worldwide and is considereda neglected disease by the World Health Organization. The etiological agents areEchinococcus tapeworms, which display elaborate developmental traits that imply acomplex control of gene expression. MicroRNAs (miRNAs), a class of small regulatoryRNAs, are involved in the regulation of many biological processes such asdevelopment and metabolism. They act through the repression of messenger RNAs(mRNAs) usually by binding to the 3? untranslated region (3?UTR). Previously, wedescribed the miRNome of several Echinococcus species and found that miRNAs arehighly expressed in all life cycle stages, suggesting an important role in geneexpression regulation. However, studying the role of miRNAs in helminth biologyremains a challenge. To develop methodology for functional analysis of miRNAs intapeworms, we performed miRNA knockdown experiments in primary cell cultures ofEchinococcus multilocularis, which mimic the development of metacestode vesiclesfrom parasite stem cells in vitro. First, we analysed the miRNA repertoire of E.multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterianmiRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs.Using genomic information and bioinformatic algorithms for miRNA binding prediction,we found a high number of potential miR-71 targets in E. multilocularis. Inhibition ofmiRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) thatblock miRNA function. To this end, we evaluated a variety of chemically modified antimiRsfor miR-71 knockdown. Electroporation of primary cells with 2?-O-methyl modifiedanti-miR-71 led to significantly reduced miR-71 levels. Transcriptomic analysesshowed that several predicted miR-71 targets were up-regulated in anti-miR-treatedprimary cells, including genes potentially involved in parasite development, hostparasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype as from controlcells and did not develop into fully mature metacestodes. These findings indicate animportant function of miR-71 in Echinococcus development and provide, for the firsttime, methodology to functionally study miRNAs in a tapeworm.