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Deciphering the role of miR-71 in Echinococcus multilocularis early development in vitro.
PLOS Neglected Tropical Diseases ( IF 3.4 ) Pub Date : 2019-12-27 , DOI: 10.1371/journal.pntd.0007932
Matías Gastón Pérez 1 , Markus Spiliotis 2 , Natalia Rego 3 , Natalia Macchiaroli 1 , Laura Kamenetzky 1 , Nancy Holroyd 4 , Marcela Alejandra Cucher 1 , Klaus Brehm 2 , Mara Cecilia Rosenzvit 1
Affiliation  

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.

中文翻译:

解读miR-71在多叶棘球oc球菌体外早期发育中的作用。

棘球co病是世界范围内的主要公共卫生问题,被世界卫生组织(WHO)视为被忽视的疾病。病原体是棘球E虫,其表现出复杂的发育特征,暗示着基因表达的复杂控制。MicroRNA(miRNA)是一类小的调节性RNA,它参与许多生物过程的调控,例如发育和代谢。它们通常通过与3'非翻译区(3'UTR)结合来抑制信使RNA(mRNA)发挥作用。以前,我们描述了几种棘球species菌物种的miRNome,发现miRNA在所有生命周期阶段都高度表达,表明在基因表达调控中的重要作用。然而,研究miRNA在蠕虫生物学中的作用仍然是一个挑战。为了开发用于tape虫中miRNA功能分析的方法,我们在多叶棘球E虫的原代细胞培养物中进行了miRNA敲除实验,该实验模仿了体外寄生虫干细胞的超囊泡囊泡的发育。首先,我们通过小的RNA-seq分析了多眼大肠杆菌原代细胞的miRNA库,发现miR-71(脊椎动物宿主中不存在的一种bilaterian miRNA)是表达最强的前五种miRNA之一。使用基因组信息和生物信息学算法进行miRNA结合预测,我们在多眼大肠杆菌中发现了大量潜在的miR-71靶标。miRNA的抑制可通过转染阻断miRNA功能的反义寡核苷酸(anti-miRs)来实现。为此,我们评估了多种化学修饰的抗miRs对miR-71的抑制作用。用2'-O-甲基修饰的抗miR-71电穿孔原代细胞可显着降低miR-71水平。转录组学分析显示,在抗miR处理的原代细胞中,几个预测的miR-71靶标上调,包括可能参与寄生虫发育,宿主寄生虫相互作用的基因,以及一些功能未知的基因。值得注意的是,沉默了miR-71的原代细胞培养物显示出与对照细胞截然不同的表型,并且没有发育成完全成熟的后代。这些发现表明miR-71在棘球chin虫的发育中具有重要作用,并首次提供了在tape虫中功能性研究miRNA的方法。转录组学分析显示,在抗miR处理的原代细胞中,几个预测的miR-71靶标上调,包括可能参与寄生虫发育,宿主寄生虫相互作用的基因,以及一些功能未知的基因。值得注意的是,miR-71沉默的原代细胞培养物表现出与对照细胞显着不同的表型,并且没有发展为完全成熟的后代。这些发现表明miR-71在棘球chin虫的发育中具有重要作用,并首次提供了在tape虫中功能性研究miRNA的方法。转录组学分析显示,在抗miR处理的原代细胞中,几个预测的miR-71靶标上调,包括可能参与寄生虫发育,宿主寄生虫相互作用的基因以及一些功能未知的基因。值得注意的是,沉默了miR-71的原代细胞培养物显示出与对照细胞截然不同的表型,并且没有发育成完全成熟的后代。这些发现表明miR-71在棘球chin虫的发育中具有重要作用,并首次提供了在tape虫中功能性研究miRNA的方法。沉默miR-71的原代细胞培养物显示出与对照细胞截然不同的表型,并且未发育成完全成熟的灭尾动物。这些发现表明miR-71在棘球chin虫的发育中具有重要作用,并首次提供了在tape虫中功能性研究miRNA的方法。沉默miR-71的原代细胞培养物显示出与对照细胞截然不同的表型,并且未发育成完全成熟的后代。这些发现表明miR-71在棘球chin虫的发育中具有重要作用,并首次提供了在tape虫中功能性研究miRNA的方法。
更新日期:2019-12-29
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