Abstract
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 elaborate 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 develop methodology for 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 in vitro. First, we 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. Using genomic information and bioinformatic algorithms for miRNA binding prediction, we found a high number of potential miR-71 targets in E. multilocularis. Inhibition of miRNAs can be achieved by transfection of antisense oligonucleotides (anti-miRs) that block miRNA function. To this end, 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 significantly reduced miR-71 levels. Transcriptomic analyses showed that several predicted miR-71 targets were up-regulated in anti-miR-treated primary cells, including genes potentially involved in parasite development, host parasite interaction, and several genes of as yet unknown function. Notably, miR-71-silenced primary cell cultures showed a strikingly different phenotype from control cells and did not develop into fully mature metacestodes. These findings indicate an important function of miR-71 in Echinococcus development and provide, for the first time, methodology to functionally study miRNAs in a tapeworm.
Highlights
Echinococcosis represents a major public health and economic issue in many countries and is considered a neglected tropical disease by the World Health Organization (WHO) [1,2]
Echinococcosis, caused by the larval stages of tapeworms of the genus Echinococcus, is a neglected disease that affects millions of people world-wide. These parasites show elaborate developmental features that rely on a complex control of gene expression. microRNAs are small molecules which have been discovered in the last decades and control gene expression in animals, plants and viruses. microRNAs are highly expressed in several tapeworms but their biological function in these parasites is unknown
Assuming that microRNAs will be important for parasite development, we analysed the function of these molecules in Echinococcus multilocularis, employing an in vitro model that mimics the first developmental transitions which occur in the human host
Summary
Echinococcosis represents a major public health and economic issue in many countries and is considered a neglected tropical disease by the World Health Organization (WHO) [1,2]. The E. multilocularis primary cell cultivation system [8], which initially contains around 80% germinative cells [5], is routinely used in developmental and immunological studies to mimic, in vitro, the transition of oncosphere-derived stem cells into metacestode vesicles [9,10,11,12] Due to their decisive role in parasite proliferation, the germinative cells are one of the most important cell miR-71 knockdown inhibits Echinococcus multilocularis early development in vitro types for the development of novel chemotherapeutics against echinococcosis [13]. Successful knockdown of platyhelminth parasite miRNAs has only been demonstrated for Schistosoma japonicum [22]
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