Identification of microRNA Genes in Three Opisthorchiids

Abstract

Background Opisthorchis felineus, O. viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose a serious threat to humans in some countries and cause opisthorchiasis/clonorchiasis. Chronic disease may lead to a risk of carcinogenesis in the biliary ducts. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at post-transcriptional level and are implicated in the regulation of various cellular processes during the parasite- host interplay. However, to date, the miRNAs of opisthorchiid flukes, in particular those essential for maintaining their complex biology and parasitic mode of existence, have not been satisfactorily described.Methodology/Principal FindingsUsing a SOLiD deep sequencing-bioinformatic approach, we identified 43 novel and 18 conserved miRNAs for O. felineus (miracidia, metacercariae and adult worms), 20 novel and 16 conserved miRNAs for O. viverrini (adult worms), and 33 novel and 18 conserved miRNAs for C. sinensis (adult worms). The analysis of the data revealed differences in the expression level of conserved miRNAs among the three species and among three the developmental stages of O. felineus. Analysis of miRNA genes revealed two gene clusters, one cluster-like region and one intronic miRNA in the genome. The presence and structure of the two gene clusters were validated using a PCR-based approach in the three flukes.ConclusionsThis study represents a comprehensive description of miRNAs in three members of the family Opistorchiidae, significantly expands our knowledge of miRNAs in multicellular parasites and provides a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites. Results of this study also provides novel resources for deeper understanding the complex parasite biology, for further research on the pathogenesis and molecular events of disease induced by the liver flukes. The present data may also facilitate the development of novel approaches for the prevention and treatment of opisthorchiasis/clonorchiasis.

Current status and perspectives of Clonorchis sinensis and clonorchiasis: epidemiology, pathogenesis, omics, prevention and control

“…Xu et al identified and cloned 6 new and 62 512 conserved adult C. sinensis miRNAs which are grouped into 284 families [ 97 ]. In another study, 33 novel and 18 conserved miRNAs were identified in C. sinensis (adult worms), including csi-miR-36b, which was not found by Xu et al [ 97 , 98 ]. The miRNAs of C. sinensis were concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates.…”

Extreme conservation of miRNA complements in Opisthorchiids

“…Altogether, in total, 1 billion small RNAseq reads of sixteen published miRNA NGS datasets for all 3 opisthorchiids [11, 12], the references genomes for O. viverrini [14], C. sinensis [15] and the draft assembly of O. felineus (Ershov et al in prep) were used. For RNAseq data we used annotations and expression levels directly from Pomanznoy et al [5].…”

Extreme conservation of miRNA complements in opisthorchiids

“…miRNAs are small non-coding RNAs that are post-transcriptional gene regulators with important roles in many biological processes [10]. Previously, miRNA complements of the 3 opisthorchiids were published and they showed a surprisingly variable number of miRNA genes (between 16 and 18 conserved and between 20 and 43 novel genes) that was unexpected [11,12]. The number of identified genes was also very low given the predictions from studies on miRNA evolution in flatworms [13] but most importantly no abundantly expressed miRNA was identified that showed sequential differences in all 3 species.…”

“…Altogether, in total, billion small RNAseq reads of sixteen published miRNA NGS datasets for all 3 opisthorchiids [11,12], the references genomes for O. viverrini [14], C. sinensis [15] and the draft assembly of O. felineus (Ershov et al in prep) were used. For RNAseq data we used annotations and expression levels directly from Pomanznoy et al [5].…”

“…For RNAseq data we used annotations and expression levels directly from Pomanznoy et al [5]. Briefly, small RNAseq reads of the projects PRJNA270708 [11] and PRJNA127731 [12] were downloaded from the Sequence Read Archive (SRA) and processed as described before [11,16]. Genomic references were downloaded from http://parasite.wormbase.org/ (PRJDA72781 & PRJNA222628) and made available by Ershov et al respectively.…”