miRNAs and lncRNAs in Echinococcus and Echinococcosis

Echinococcosis are considered to be potentially lethal zoonotic diseases that cause serious damage to hosts. The metacestode of Echinococcus multilocularis and E. granulosus can result in causing the alveolar and cystic echinococcoses, respectively. Recent studies have shown that non-coding RNAs are widely expressed in Echinococcus spp. and hosts. In this review, the two main types of non-coding RNAs—long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)—and the wide-scale involvement of these molecules in these parasites and their hosts were discussed. The expression pattern of miRNAs in Echinococcus spp. is species- and developmental stage-specific. Furthermore, common miRNAs were detected in three Echinococcus spp. and their intermediate hosts. Here, we primarily focus on recent insights from transcriptome studies, the expression patterns of miRNAs and lncRNAs, and miRNA-related databases and techniques that are used to investigate miRNAs in Echinococcus and echinococcosis. This review provides new avenues for screening therapeutic and diagnostic markers.


Introduction
The metacestode form of Echinococcus spp. cestode parasites can result in the echinococcosis in the visceral organs (such as the liver and lung) of intermediate hosts. E. granulosus sensu lato (s.l.) and E. multilocularis are the two of the most common and researched harmful parasites [1]. Worldwide zoonoses are of great concern to public health; echinococcosis occurs worldwide and yet the amplitude of its severity is thought to be overlooked by the World Health Organization [1] and indeed occurs worldwide [2]. Cystic echinococcosis (CE) is typically the result of accidental ingestion of E. granulosus eggs and initiating detrimental effects to the liver and lungs [3] ( Figure 1A). Mature adult E. granulosus tapeworms were found in the small intestine of the definitive host, a carnivore, are secreted in faecal matter releasing segments, or proglottids, comprised of large quantities of eggs, contaminating proximate vegetation and water sources. The larva then penetrate the intestinal wall of the intermediate hosts (such as sheep) and migrate through the circulation to various throughout the hosts body, in most cases, the liver and lungs. Whole cysts of E. granulosus are comprised of a cyst wall (CW) and hydatid cyst fluid. The brood capsules, protoscoleces, and free daughter cysts in hydatid cyst fluid are collectively referred to as hydatid sand [4]. Cystic echinococcosis can be characterised by the long-term growth of hydatid cysts in mammalian intermediate hosts and humans, imposing cystic echinococcosis [19]. In addition, lncRNAs have also been identified as important in the biological functions of other parasites (e.g., Trichomonas vaginalis [36] and Toxoplasma [37]) and in host immunity. Thus, we speculate that lncRNAs may be expressed in Echinococcus spp. and perform large-scale biological functions in both parasites and hosts.  [38,39]. The mature miRNA can be loaded into the RNA-induced silencing complex (RISC), resulting of miRNA, Dicer, the RNA-binding protein Argonaute (AGO), and the adaptor protein TAR-RNA-binding protein (TRBP) [40]. The complementary sequences in the untranslated regions of lncRNAs, mRNAs, circRNAs, and pseudogenes can competitively bind to miRNAs, which results in translational repression and degradation of mRNA and miRNA cleavage. (2) CircRNA: circRNAs are spliced and transcribed from genomic DNA and transported to the cytoplasm to perform numerous biological functions. One example of such functions is the translation of derived pseudogenes, sponge proteins and miRNAs, into polypeptides [41]. (3) LncRNA: lncRNAs play important biological roles in the cytoplasm; including acting as signalling molecules, decoys, guides, and scaffolds; being translated into polypeptides, and serving as sources of small interfering RNAs (siRNAs), miRNAs, and Piwi-interacting RNAs (piRNAs) [42].

MiRNAs Expressed in Different Developmental Stages of E. granulosus Sensu Stricto
MiR-2, miR-71, and miR-125 have the highest expression levels among the 76 known miRNAs of E. granulosus sensu stricto [44,45]. Interestingly, the expression levels of miR-124b* and miR-87* are higher than those of their mature miRNAs, which suggests that they act as effectors during development and derivatives of their corresponding pre-miRNAs produce two different regulatory small RNAs [45]. In addition, miRNAs were found to exhibit tissue-and phase-specific expression [45]. MiR-277, let-7, miR-71, miR-10, miR-2, and miR-9 are specifically expressed in the cysts walls of secondary hydatid cyst and protoscoleces of G1 and G7 genotype, whereas miR-125 is only detected in protoscoleces and pre-microcysts. Additionally, three miRNAs (let-7, miR-71, and miR-2) are expressed at high levels in protoscoleces of metacestodes (cyst walls), which suggests that their expression is developmentally regulated [45]. Gene Ontology (GO) enrichment analysis revealed that the differentially expressed miRNAs in E. granulosus and their potential targets may participate in nutrient metabolism and bi-directional development of the nervous system [44].

Common miRNAs in Echinococcus spp.
In this review, all of the identified miRNAs in E. granulosus sensu stricto, E. multilocularis and E. canadensis (G7) were obtained from the reported articles [1,12,[44][45][46]. Across the three species analysed, some miRNAs were shown to be highly conserved, which suggested further functional conservation. Eighty-seven miRNAs, suggesting highly conserved miRNAs may perform crucial roles in the development and parasitism of Echinococcus spp. Among these highly conserved miRNAs; miR-71, bantam, let-7, miR-9, miR-10, miR-7, miR-87, and miR-61 were the most highly expressed miRNAs in E. multilocularis and E. Canadensis (G7) infection of the intermediate host [1,12,44]. The function of the miRNAs miR-71, let-7, and miR-61 were verified [44,49], and the target of miR-71 was also confirmed ( Table 2), in total, the targets and biological functions of fifteen miRNAs in Echinococcus were predicted. The miRNA let-7 exhibited a substantially increased expression in protoscoleces and cysts, which might be associated with the bi-directional development capabilities of E. granulosus [44]. Moreover, Mortezaei et al. demonstrated that, under benzimidazole exposure in vitro, the expression of E. granulosus miRNAs let-7 and miR-61 was significantly affected in the microcyst stage; however, these miRNAs exhibited different alteration patterns in response to albendazole sulfoxide in other developmental stages [49]. In addition, the ubiquitin-conjugating enzyme E2 was identified as the potential target of miR-307, suggesting that miR-307 might be involved in ubiquitin-mediated proteolysis and herpes simplex infection signalling pathways in Echinococcus [23].
MiRNAs highly expressed in Echinococcus, but not expressed in vertebrate host, may have diverged from their host homologue miRNAs, for instance, bantam, miR-71, and miR-277 [12], can be assessed as candidate targets for diagnostic markers and intervention strategies. A previous study found that nematode exosome-derived miR-71 plays an important role interaction between the host and parasite, as an innate immune regulator [50,51]. A mimic of Echinococcus-derived miR-71 did not change the level of IL-10 in mouse RAW264.7 cells to evade host immune surveillance [52,53]. Furthermore, it can also be deduced that miR-277 might be involved in regulating Wnt signalling pathways, which are responsible for the regulation of stem cell pluripotency in Echinococcus [23]. The conservation of miRNAs that are involved in Echinococcus regulation reflects the complex and sophisticated adaptations, which are necessary for different environments, present within the life cycles of parasitic species.

Non-Coding RNAs in Intermediate Hosts during Infection with Echinococcus spp.
High-throughput sequencing and miRNA microarray analyses identified dysregulated miRNAs present during parasite infection in natural hosts and animal models that were present in relevant cells, tissues, and blood; demonstrating the importance of these miRNAs in host responses to pathogen challenges [19,43,55]. Recent research results have shown that circulating non-coding RNAs, including miRNAs and lncRNAs, can be stably detected in the blood of hosts that were infected with E. granulosus and E. multilocularis [19,43,56] (Table 3). These stably circulating non-coding RNAs have the potential to provide us with an understanding of their roles in the host-parasite interaction, development, and growth; and, could potentially serve as diagnostic targets and therapeutic candidates.

MiRNAs and lncRNAs in Host Responses to E. granulosus
MiRNA and lncRNA profiles change when hosts are infected with E. granulosus ( Figure 2). The gut of the intermediate host is integral to the process, as the first effector of host defence against Echinococcus spp. Sheep are highly susceptible to cystic echinococcosis as intermediate hosts. NF-κB pathway-responsive miRNAs, which are related to the inflammation process, are expressed in significantly higher proportions in CE-resistant sheep than in non-CE-resistant sheep, specifically miR-27a, miR-542-5p, miR-134-5p, miR-21-3p, miR-26b, and miR-671 [57] (Figure 2). It could therefore be concluded from aforementioned results that the differential expression of miRNAs present in CE-resistant and non-CE-resistant sheep may be key in the response of intestinal tissues to E. granulosus. Myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of myeloid cells, are composed of dendritic cells, granulocytes, and terminally differentiated macrophages; parasitic infection results in aberrant MDSC expansion [58]. MDSCs accumulate to high levels in mouse models [19,59] and they have demonstrated an important function in the down regulation of the immune response of T lymphocytes when infected with E. granulosus protoscoleces. Several differentially expressed lncRNAs and mRNAs were identified between the normal mice and splenic monocytic MDSCs of E. granulosus protoscoleces-infected mice [19] (Figure 2). KEGG pathway enrichment analysis suggests that the lncRNAs co-expressed with mRNAs are mainly primarily involved in regulating the vascular endothelial growth factor (VEGF) signalling pathway, the leishmaniasis, Salmonella infection, and actin cytoskeleton [19]. The results showed that the aforementioned transcription factors are known to regulate lncRNA production, several of the most likely transcription factors (PGR, IL6, YY1, and FOSL1) for those lncRNAs were predicted by lncRNA-target-transcription factor network analysis [19]. These transcription factors mainly regulate the lncRNAs FR049933, FR291292, FR110455, and FR400826 and they participate in the MAPK and VEGF signalling pathways that are involved in MDSC function [19]. Specifically, the retinoblastoma gene Rb1, the expression of which is associated with abnormal M-MDSC differentiation, and was cis-regulated by the lncRNA NONMMUT021591 [19]. Such results show that lncRNAs participate in the immune regulation of the intermediate host, mice, in their defence against E. granulosus, and might be useful as specific biomarkers for CE.

Mouse miRNAs Dysregulated during Infection with E. multilocularis
The miRNA expression levels of mice were found to significantly in the sera and livers from mice in different stages of E. multilocularis infection [43,60] (Figure 2). Mmu-miR-146a-5p, mmu-miR-107-3p, mmu-miR-103-3p, and mmu-miR-21a-3p were found to be significantly upregulated after four weeks of infection. Furthermore, the expression of mmu-miR-339-5p was significantly upregulated at four weeks post-infection, but did not differ from baseline at eight or 12 weeks post-infection. In contrast, mmu-miR-222-3p was found to be significantly downregulated throughout the process of infection. The infectious stage of E. multilocularis can be estimated by the expression levels of these miRNAs. The GO terms enriched in potential miRNA targets are involved in the metabolism, signal transduction, immune response, and gene expression regulation [43]. Among E. multilocularis-derived circulating miRNAs, only emu-miR-10, emu-miR-227, and emu-miR-71 have been verified [43].
These E. multilocularis-derived circulating miRNAs may be used as potential diagnostic markers in intermediate hosts. The levels of three miRNAs (mmu-miR-378a-3p, mmu-miR-101b-3p, and mmu-miR-192-5p) were significantly decreased 90 days post-inoculation when compared to 30 days post-inoculation in mouse livers [60]. These results can inform further studies of the role of host miRNAs during E. multilocularis infection.

Common miRNA Families in the Host Model during Infection with Echinococcus spp.
All of the identified miRNAs in the sheep gut and in mouse macrophages, livers, and sera were collected from published literature [43,57,60]. The confirmed miRNA families were then classified according to their annotations in these articles [43,57,60]. Twenty-two common miRNA families were distinguished in intermediate hosts (including sheep and mice) during infection with Echinococcus spp. (Table 3), all of which were upregulated in the sheep gut and showed differential expression levels in mouse macrophages, livers, and sera. MiRNAs (e.g., miR-181 [61], miR-30 [62], miR-365 [63], miR-378 [64], miR-449 [65], miR-99 [66], miR-130 [67], and miR-16 [68]) have multiple target genes, including mRNAs, lncRNAs, and circRNAs; this may be key in determining the common miRNAs involved in the hosts response to infection with Echinococcus spp., which exhibit different expression levels, functions, and targets in host sheep and mouse models. Many miRNAs, such as miR-181 [57], miR-21 [69], and miR-27 [70], were implemented in the regulation of the immune response in intermediate hosts that were infected with Echinococcus spp. Further research should focus on the functional mechanisms of these common miRNAs in hosts that were infected with Echinococcus spp. and their potential roles in the treatment of echinococcosis. MiR-181 Influences the differentiation of T helper cells and the activation of macrophages, controls T cell sensitivity to antigens during development [73] MiR-18 ↑ --↑ Unknown As the female immunity regulator, miR-18 controls the expression of A20/Tnfaip3 and exacerbating NF-κB-driven inflammation in fibroblast-like synoviocytes of rheumatoid arthritis [74] MiR-20 ↑ -↓ -ATG10 Inhibits autophagy and chondrocyte proliferation by targeting ATG10 through the PI3K/AKT/mTOR signalling pathway. [75] MiR-21 ↑ ↑ -↑

MiRNAs Mainly Associated with Immune and Pathological Processes during Host Infection with Echinococcus spp.
The functions and mechanisms of several miRNAs, such as miR-19b (E. granulosus) [91], miR-71 (E. multilocularis) [92], and miRNA-222-3p (E. multilocularis) [93], have been identified. These miRNAs can be used as potential diagnostic markers during infection with Echinococcus spp. In this section, we describe the mechanisms and potential uses of these miRNAs in the diagnosis and treatment of echinococcosis.

MiR-71 as an Innate Immune Regulator in Echinococcosis
Extensive research of miR-71 has been conduced, in particular concerning Echinococcus and echinococcosis, revealing that miR-71 is a conserved miRNA that is widely expressed in parasites. Nematode exosome-derived miR-71 can be internalized by host cells and serve as an innate immune regulator [50], performing a significant role in host-parasite interactions [51]. MiR-71 also functions in E. multilocularis protoscolex development, in which it is differentially expressed at various developmental stages and is found at higher levels in protoscoles without hooks than those with hooks [54]. The Nemo-like kinase gene (nlk) is the target of miR-71, when miR-71 binds with nlk, NLK expression is inhibited [54]. Thus, miR-71 might play an integral part in the development of alveolar Echinococcus [92]. These results lay the groundwork for further exploration into new drugs acting through miR-71 and nlk to treat alveolar echinococcosis. Alveolar Echinococcus-derived miR-71 also participates in regulating the immune process in mouse macrophages [53]. MiR-71 mimic-transfected RAW264.7 cells do not show significantly altered levels of IL-10 when compared with negative control-transfected RAW264.7 cells, which exhibit significantly repressed NO production at 12 h post-treatment [52]. NO is involved in affecting immunosuppressive anti-parasite immune responses and limiting parasite infection, suggesting that it has essential roles in early and chronic infections of Echinococcus spp. [94]. Some components and molecules, such as crude parasite extracts, a laminated layer, and 14-3-3 proteins, have been identified as being able to inhibit NO release by macrophages [94]. Host macrophages may take up parasite-derived miR-71 that is released into the host microenvironments, body fluids, serum, and plasma. Thus, miR-71 is involved in the regulation of Echinococcus spp. development and function in host macrophages and can be useful for studying host-parasite interactions.

miR-19b as an Effective Treatment Biomarker
A previous study described that miR-19b plays a part in various pathological conditions and diseases, such as fibrogenesis, osteosarcoma, and clear cell renal cell carcinoma [91,[95][96][97]. In cystic echinococcosis, peri-cystic fibrosis is accelerated by hydatid cyst fluid [98]. The expression level of miR-19 is downregulated during the progression of hepatic stellate cell (HSC) activation and, similarly, it is significantly decreased in the patients liver tissues with cystic echinococcosis, and this was also found to be the case in a mouse model of liver fibrosis [98]. Furthermore, miR-19b expression was found to be significantly downregulated in fibrotic liver samples when compared to that in neighboring normal liver tissues, interestingly COL1A1 mRNA expression showed significant-negative correlations with the expression of miR-19b [98]. Hydatid cyst fluid significantly promotes the proliferation of LX-2 cells by accelerating the transition from G0/G1 phase to S phase, increasing the mRNA and protein expression levels of COL3A1, TGFβRII, COL1A1, and α-SMA, [98]. MiR-19b overexpression in hydatid cyst fluid-treated LX-2 cells leads to the significant suppression of cell proliferation and decreases in TβRII, COL1A1 and COL3A mRNA, and protein expression levels by blocking signal transmission in the TGF-β pathway delaying, or potentially reversing the progression of fibrosis [98]. Thus, hydatid cyst fluid is involved in the progression of fibrosis via the activation of hepatic stellate cells, and the regulation of miR-19 expression is sectional of the mechanism regulating peri-cystic fibrogenesis in cystic echinococcosis. Previous studies have demonstrated that TGF-β/Smad pathway activation is the consequence of infections by E. multilocularis [99,100]; the activation of this pathway impacts host-parasite interactions, such as fibrogenesis, hepatic (and possibly metacestode) cell proliferation, and immune tolerance mechanisms [98]. These results suggest that E. granulosus can promote fibrosis and restrain liver miR-19 expression by increasing TβRII expression, extracellular matrix production, and activating hepatic stellate cells [98]. Furthermore, these results provide new evidence supporting the involvement of miRNAs in regulating fibrosis in infectious diseases. The overexpression of miR-19 in the liver might be an effective treatment biomarker in intermediate hosts that were infected with E. granulosus.

miR-222-3p Modulates Macrophage Immunity
Studies have reported that miR-222-3p is implicated in the regulation of vascular physiology and many malignant inflammatory diseases [93]. During infection, E. multilocularis has been shown to dysregulate the expression of miRNAs in the liver and serum of infected mice [43,60]. For example, mouse miR-222-3p tends to be downregulated and significantly decreased at two months and three months post-infection, respectively, in the spleens of infected mice as compared with control mice [43]. Furthermore, crude E. multilocularis antigens significantly inhibit miR-222-3p expression [6]. Macrophages that were transfected with miR-222-3p inhibitors have been shown to moderately decrease NO secretion, relative to control macrophages. Analysis of transfected cells revealed four key genes implicated in the LPS/TLR4 signalling pathway were found to be significantly down-or unregulated; of which TICAM2, TLR4, and CD14 were upregulated, while AP1 was downregulated [6]. Therefore, miR-222-3p downregulation can modulate macrophage immune functions by regulating NO secretion and the LPS/TLR4 signalling pathway, which potentially contributes to the pathogenesis of alveolar echinococcosis. Thus, miR-222-3p downregulation might be useful as an auxiliary diagnostic marker for alveolar echinococcosis.

Echinococcus miRNA-Related Databases
Various datasets and software programs have been utilized for predicting and analyzing miRNAs in multiple species. Numerous Echinococcus miRNAs have been identified via various transcriptomic and modern computational approaches. The application of these databases and software programs could effectively accelerate the exploration of Echinococcus miRNA functions and mechanisms ( Table 4). The Wellcome Sanger Institute and Sequence Read Archive provide raw genome and miRNA sequencing data, respectively (Chinese Human Genome Centre at Shanghai, and Trust Sanger Institute) [12,14]. Echinococcus miRNAs can be authenticated with miRBase [101] and Rfam [102]. Furthermore, miRanda [103], RNA22 [104], RNAhybrid [105], and TarBase v6.0 [106] have been used for predicting and evaluating the targets of miRNAs.

Techniques and Methods Used in miRNA Studies in Echinococcus and Echinococcosis
MiRNA studies in Echinococcus and echinococcosis have primarily explored miRNA identification, functions, and mechanisms. Numerous sequencing, bioinformatic analyses, and experimental verification techniques have been utilized to precisely characterize miRNA expression profiles and function mechanisms.

MiRNA Identification
Sanger sequencing [45], modern sequencing techniques (such as RNA sequencing (RNA-seq)) [1,12,44], and bioinformatic analysis approaches (e.g., self-organizing map analysis) [46] have been used to identify miRNAs in Echinococcus and echinococcosis. Among these methods, modern techniques (e.g., the Illumina Genome Analyzer II for small RNA sequencing) provide a high-throughput approach for the large-scale detection of miRNA expression in Echinococcus and echinococcosis. The validity of the sequencing results and miRNA expression levels can be subsequently verified via Northern blotting [45] and qRT-PCR [12,60]. Bioinformatic analysis, an accurate and convenient approach, is an effective tool that can be used for further verification of new and known miRNAs [46]. For example, a novel deep architecture of SOMs was used to predict novel miRNAs that are based on the complete genome of E. multilocularis without the need for RNA-seq data or target analysis for prediction. In theory, this methodology can be easily adapted and applied to any draft genome [46]. However, in practice, these techniques have some limitations. Sanger sequencing is a first-generation sequencing technique, however it is unsuitable for large-scale sequencing. QRT-PCR demands more advanced experimental techniques, but it has the discommodity of high cost and low throughput. The development of high-throughput sequencing technology and the continual expansion of genome libraries has ushered in recent bioinformatic approaches for miRNA discovery (such as the Megablast algorithm and SOM analysis). Despite these advancements, bioinformatic analyses based on big data can yield false positive results, thus further advancements needs to be made to improve the accuracy of experimental tests and provide the necessary verification for the results of bioinformatic analyses.

Verification of the Functions and Exploration of the Mechanisms
Although many Echinococcus and echinococcosis-related miRNAs have been identified, the functions and mechanisms of action of most are unclear. Loss-of-function (LOF) and gain-of-function (GOF) studies are usually employed to examine gene functions [110]. The in vivo study of miRNA functions in Echinococcus proves difficult under the current achievable experimental conditions. Nonetheless, specific miRNAs from Echinococcus hosts, including miRNAs from Echinococcus itself, have been identified. LOF studies of echinococcosis-related miRNAs can be carried out in host cells, such as hepatic stellate cells [98] and mouse macrophages [53], with inhibitors or siRNAs [6]. In vitro GOF studies of echinococcosis-related miRNAs in host cells have been performed via the transfection of gene mimics [98]. Additionally, crude Echinococcus antigens have been used to treat host cells to study miRNAs and changes in the expression levels of their targets [6].

Conclusions
To date, a series of miRNAs have been identified in Echinococcus spp., but the function and mechanism of most have not been validated. More advanced methods need to be applied to identify effective miRNAs and their functions to deeply understand parasite physiology and to screen for useful diagnostic and treatment targets. As one of the most important hosts of Echinococcus spp. and due to the inadvertent nature of infection to humans, more sensitive and discriminatory diagnostic indicators are needed for human echinococcosis in the early stage of infection. Although recent studies have concentrated on miRNAs in Echinococcus spp. metacestodes, more research should be conducted on non-coding RNAs in adult cestodes and definitive hosts. Several reported functional miRNAs, such as miR-71, miR-19b, and miR-222-3p, have potential applications in the study of host-parasite interactions and as treatment targets in echinococcosis, therefore they should receive the increased attention. However, despite their potential, clinical application of these functional miRNAs is distant. Moreover, the function and mechanism of action of many identified miRNAs remain unknown. Future research should devote ample attention to screening for miRNA-based early diagnostic markers and treatment targets for echinococcosis in hosts.
In addition to miRNAs, other non-coding RNAs, such as lncRNAs and circRNAs, may play regulatory roles in Echinococcus spp. and echinococcosis. Recently, the competing endogenous effect has contributed to our understanding of miRNA regulatory mechanisms at the post-transcriptional level. Protein-coding RNAs, lncRNAs, pseudogenes, and circRNAs act as miRNA sponges. Furthermore, these miRNA sponges interact with each other through shared miRNAs and participate in crosstalk to develop miRNA-mediated interactions or miRNA sponge interaction networks. Therefore, identifying lncRNAs and circRNAs in Echinococcus spp. and echinococcosis is necessary for providing new targets for potential treatment and diagnosis.