Pérez M.G., Spiliotis M., Rego N., Macchiarioli N., Kamenetzky L., Holroyd N., Cucher M.A., Brehm K. and Rosenzvit M.C.
PLoS Neglected Tropical Diseases 2019
Echinococcosis represents a major public health problem worldwide and is considered a neglected disease by the World Health Organization. The etiological agents are Echinococcus tapeworms, which display particular developmental traits that imply a complex control of gene expression. MicroRNAs (miRNAs), a class of small regulatory RNAs, are involved in the regulation of many biological processes such as development and metabolism. They act through the repression of messenger RNAs (mRNAs) usually by binding to the 3’ untranslated region (3’UTR). Previously, we described the miRNome of several Echinococcus species and found that miRNAs are highly expressed in all life cycle stages, suggesting an important role in gene expression regulation. However, studying the role of miRNAs in helminth biology remains a challenge. To provide a means of functional analysis of miRNAs in tapeworms, we performed miRNA knockdown experiments in primary cell cultures of Echinococcus multilocularis , which mimic the development of metacestode vesicles from parasite stem cells. We first analysed the miRNA repertoire of E. multilocularis primary cells by small RNA-seq and found that miR-71, a bilaterian miRNA absent in vertebrate hosts, is one of the top five most expressed miRNAs. Then we predicted miR-71 mRNA targets using genomic information and observed a high number of predicted targets. The inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. Here we evaluated a variety of chemically modified anti-miRs for miR-71 knockdown. Electroporation of primary cells with 2’-O-methyl modified anti-miR-71 led to a significant reduction of miR-71 levels. Transcriptomic analyses showed that several predicted miR- 71 targets were up-regulated in anti-miR-treated primary cells, including genes involved in parasite development such as a frizzled ortholog, a GPCR receptor presumably acting in the Wnt signaling pathway, and genes involved in parasite-host interplay such as EmTIP, as well as genes coding for tapeworm-specific proteins of unknown function. Notably, miR-71-silenced primary cells showed a different phenotype from control cells and did not develop metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide methodology for miRNA functional analysis in this parasite that could be applied to related tapeworms.
The associated script and data files enable the reproduction of the DESeq2 analysis explained under the manuscript subtitle “Library construction, sequencing and bioinformatics analyses of RNA-seq data after knockdown experiments”.