miRNA BIOGENESIS

Primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase II or III (Cai et al. Reference Cai, Hagedorn and Cullen2004; Borchert et al. Reference Borchert, Lanier and Davidson2006). The pri-miRNA is then processed by a nuclear microprocessor complex, composed of Drosha (a RNase III enzyme) and DGCR8 (DiGeorge critical region 8, also known as Pasha in Drosophila melanogaster and C. elegans), into a precursor-miRNA (pre-miRNA) that is a stem-loop hairpin precursor (Lee et al. Reference Lee, Ahn, Han, Choi, Kim, Yim, Lee, Provost, Radmark, Kim and Kim2003, Reference Lee, Kim, Han, Yeom, Lee, Baek and Kim2004). The pre-miRNA is then exported from the nucleus to the cytoplasm through exportin-5 in a Ran-GTP- and dsRNA-dependent manner, where it is further processed into a short duplex of 20–24nts by Dicer (another RNase III enzyme). Subsequently, a single strand from this duplex is preferentially loaded into the RNA Induced Silencing Complex (RISC) whose core binding protein is a member of the Argonaute protein family (Flynt and Lai, Reference Flynt and Lai2008). The miRNA/RISC binds directly to the 3′ UTR, 5′UTR or the open reading frame of the mRNAs through partial or complete complementary pairing between mRNA and miRNA sequences, typically resulting in mRNA degradation and/or protein translation inhibition (Moretti et al. Reference Moretti, Thermann and Hentze2010; Humphreys et al. Reference Humphreys, Westman, Martin and Preiss2005; Filipowicz et al. Reference Filipowicz, Bhattacharyya and Sonenberg2008; Qin et al. Reference Qin, Shi, Zhao, Yao, Jin, Ma and Jin2010). Recently, miRNAs have also been shown to be involved in translational activation (Filipowicz et al. Reference Filipowicz, Bhattacharyya and Sonenberg2008) and heterochromatin formation (Benetti et al. Reference Benetti, Gonzalo, Jaco, Munoz, Gonzalez, Schoeftner, Murchison, Andl, Chen, Klatt, Li, Serrano, Millar, Hannon and Blasco2008; Kim et al. Reference Kim, Saetrom, Snove and Rossi2008). Through these actions, miRNAs regulate gene expression during development, differentiation, proliferation, cell apoptosis and death, and metabolism in many organisms (Bushati and Cohen, Reference Bushati and Cohen2007). However, not all miRNAs are processed by this so-called canonical biogenesis pathway. Alternatively, some miRNAs are spliced and debranched from short intronic hairpins that bypass cleavage of Drosha, but nuclear export and further processing are common with the canonical miRNA pathway (Okamura et al. Reference Okamura, Hagen, Duan, Tyler and Lai2007; Ruby et al. Reference Ruby, Jan and Bartel2007).

Most miRNAs are under the control of developmental and/or tissue-specific signals and precise regulation of miRNA expression is crucial to maintain normal cellular functions. miRNA expression can be regulated both at the transcriptional level and at the post-transcriptional level. Many cellular transcription factors including c-Myc, p53, and E2F have been described to regulate miRNA transcription (He et al. Reference He, He, Lim, de Stanchina, Xuan, Liang, Xue, Zender, Magnus, Ridzon, Jackson, Linsley, Chen, Lowe, Cleary and Hannon2007; Woods et al. Reference Woods, Thomson and Hammond2007). Protein factors that interact with Drosha, Dicer and the terminal loop of pri- or pre-miRNAs can also regulate miRNA biogenesis. For example, p68/p72 enhances or represses maturation of a subset of miRNAs (94 out of 266 miRNAs investigated; Fukuda et al. Reference Fukuda, Yamagata, Fujiyama, Matsumoto, Koshida, Yoshimura, Mihara, Naitou, Endoh, Nakamura, Akimoto, Yamamoto, Katagiri, Foulds, Takezawa, Kitagawa, Takeyama, O'Malley and Kato2007). The KH-type splicing regulatory protein (KSRP) (Trabucchi et al. Reference Trabucchi, Briata, Garcia-Mayoral, Haase, Filipowicz, Ramos, Gherzi and Rosenfeld2009) and Lin28 (Viswanathan et al. Reference Viswanathan, Daley and Gregory2008), the specific interactors for miRNA precursor loops between RNA-binding proteins and cis-regulatory sequences, regulate the biogenesis of a subset of miRNAs.

SCHISTOSOME miRNA PATHWAY AND miRNA DISCOVERY

Schistosome miRNA pathway

The miRNA pathway consists of a conserved core of proteins and enzymes found in human, C. elegans, Drosophila melanogaster and Arabidopsis thaliana and known to function in the recognition and processing of miRNAs. Based on the most up-to-date model of miRNA biogenesis and regulated processing, bioinformatic analyses suggest that the schistosome genome encodes most of the known core factors and enzymes involved in miRNA biogenesis (Table 1). In S. mansoni, 13 putative proteins homologous with the known enzymes involved in the miRNA pathway in other organisms, including Dicer, Drosha, Ago and exportin-5, have been predicted using comparative genomics (Gomes et al. Reference Gomes, Cabral, Jannotti-Passos, Carvalho, Rodrigues, Baba and Sa2009). Since Ago proteins are the primary mediators of miRNA function, we identified three Ago proteins in S. japonicum and examined their expression profiles during schistosome development. Both S. japonicum eggs and miracidia showed relatively high expression of all three Ago proteins (Chen et al. Reference Chen, Yang, Guo, Peng, Liu, Li, Lin and Cheng2010b), a result suggesting that Ago proteins and/or their coordinated miRNAs may regulate gene expression and then control schistosome development and other biological processes (Chen et al. Reference Chen, Guo, Yang, Liu, Lin, Li and Cheng2010a, Reference Chen, Yang, Guo, Peng, Liu, Li, Lin and Chengb; Yang et al. Reference Yang, Guo, Chen, Lin, Liu and Cheng2010).

Table 1. Putative proteins involved in miRNA biogenesis and its regulation in schistosomes

* These putative proteins were also reported by Gomes (Gomes et al. Reference Gomes, Cabral, Jannotti-Passos, Carvalho, Rodrigues, Baba and Sa2009).

Schistosome miRNA discovery

To date, several groups have carried out the studies to identify miRNAs in schistosomes, particularly in S. japonicum (Table 2), and to provide some insight into the role of miRNAs in schistosome development.

Table 2. Summary of miRNA discovery in schistosomes

* A, Adult worms; C, Cercaria; S, Schistosomula.

To the best of our knowledge, Xue and co-workers first reported the identification of miRNAs in S. japonicum (Xue et al. Reference Xue, Sun, Zhang, Wang, Huang and Pan2008). They identified five miRNAs including Sja-let-7, Sja-miR-71, Sja-bantam, Sja-miR-125 and Sja-miR-new1 in S. japonicum by direct cloning. They reported that four out of five miRNAs were shown to be conserved in other organisms and that the miRNA expression profiles were highly stage-specific (Xue et al. Reference Xue, Sun, Zhang, Wang, Huang and Pan2008). In a more comprehensive study, Wang and co-workers used a high-throughput sequencing technology, Illumina Solexa, to characterize small RNAs populations in S. japonicum schistosomula, the early stage in the vertebrate host. They identified 20 evolutionarily conserved miRNAs and 16 schistosome-specific miRNAs (Wang et al. Reference Wang, Xue, Sun, Luo, Xu, Jiang, Zhang and Pan2010). Using a similar approach, Huang et al. (Reference Huang, Hao, Chen, Hu, Yan, Liu and Han2009) reported 176 species-specific miRNAs in immature schistosomula and mature, paired adult S. japonicum while Hao and co-workers identified 16 evolutionarily conserved miRNAs and 22 schistosome-specific miRNAs (Hao et al. Reference Hao, Cai, Jiang, Wang and Chen2010) in the same organism. In S. mansoni, Simoes et al. (Reference Simoes, Lee, Djikeng, Cerqueira, Zerlotini, da Silva-Pereira, Dalby, LoVerde, El-Sayed and Oliveira2011) identified 211 novel miRNA candidates by sequencing small RNA cDNA libraries from adult worm pairs and presented data supporting stage-regulated expression patterns for some of the miRNAs.

Although the Illumina deep sequencing has been applied for schistosome miRNA discovery in several studies, the number of miRNAs identified showed significant variation between these studies (Table 2). Differing starting material and procedures used for RNA isolation, library construction and/or sequencing can affect the number of miRNAs identified. The criteria used to define a miRNA in these studies were different and less stringent than those currently used for high-confidence miRNA annotation (Meyers et al. Reference Meyers, Axtell, Bartel, Bartel, Baulcombe, Bowman, Cao, Carrington, Chen, Green, Griffiths-Jones, Jacobsen, Mallory, Martienssen, Poethig, Qi, Vaucheret, Voinnet, Watanabe, Weigel and Zhu2008; Berezikov et al. Reference Berezikov, Liu, Flynt, Hodges, Rooks, Hannon and Lai2010; Chiang et al. Reference Chiang, Schoenfeld, Ruby, Auyeung, Spies, Baek, Johnston, Russ, Luo, Babiarz, Blelloch, Schroth, Nusbaum and Bartel2010). For high-confidence miRNA annotation, apart from the requirement that the miRNA should be derived from a unique and relatively stable genomic hairpin predicted by bioinformatics, the following criteria should be also jointly considered: (1) miRNAs have multiple cloned 21-24nt reads with relatively fixed 5′ ends; (2) the miRNA* (an miRNA* denotes the small RNA processed from the hairpin arm opposite the mature miRNA) is also presented in the reads; (3) the miRNA and miRNA* form a duplex with two nucleotide, 3′ overhangs; (4) with sufficient sequencing, it is also possible to present other byproducts of miRNA biogenesis such as terminal loops or species flanking the pre-miRNA hairpin. The defined miRNAs in schistosomes will be narrowed significantly when these combined criteria are applied. Nevertheless, these studies have still provided substantial evidence to support the view that miRNAs serve as critical regulators in the regulation of gene expression in schistosomes and also imply miRNA roles in schistosome development.

EVOLUTIONARILY CONSERVED miRNAS IN SCHISTOSOMES

Many miRNAs from bilateral animals are phylogenetically conserved; 55% of C. elegans miRNAs have homologues in humans. This suggests that miRNAs have played important roles throughout animal evolution (Pasquinelli et al. Reference Pasquinelli, Reinhart, Slack, Martindale, Kuroda, Maller, Hayward, Ball, Degnan, Muller, Spring, Srinivasan, Fishman, Finnerty, Corbo, Levine, Leahy, Davidson and Ruvkun2000; Ibanez-Ventoso et al. Reference Ibanez-Ventoso, Vora and Driscoll2008). Some conserved miRNAs are involved in critical biological processes such as cell differentiation and proliferation, apoptosis, metabolism and development (Reinhart et al. Reference Reinhart, Slack, Basson, Pasquinelli, Bettinger, Rougvie, Horvitz and Ruvkun2000; Johnson et al. Reference Johnson, Grosshans, Shingara, Byrom, Jarvis, Cheng, Labourier, Reinert, Brown and Slack2005; Chen et al. Reference Chen, Mandel, Thomson, Wu, Callis, Hammond, Conlon and Wang2006; Caygill and Johnston, Reference Caygill and Johnston2008). Furthermore, it has been demonstrated that an evolutionarily conserved miRNA coevolves with its target mRNA, retaining a similar development or physiological role in diverse taxa (Lee and Ambros, Reference Lee and Ambros2001; Takane et al. Reference Takane, Fujishima, Watanabe, Sato, Saito, Tomita and Kanai2010).

Based on the conserved miRNAs reported by individual studies, we selected S. japonicum miRNAs that have been identified in several independent studies and examined their evolutionary conservation in humans (Homo sapiens), Mus musculus, D. melanogaster, C. elegans and a member of the same phylum (Platyhleminthes) as schistosomes, the fresh-water planarian Schmidtea mediterranea. Briefly, S. japonicum miRNAs were searched against the miRbase by using BLASTN (http://www.mirbase.org) at the default setting and then the pairs were subjected to manual checks. As summarized in Table 3, thirteen S. japonicum miRNAs have corresponding homologues in at least three species out of five investigated (Table 3). Five miRNAs including miR-1b, miR-124, miR-8/200b, miR-190 and miR-7 are conserved among all five species (Table 3).

Table 3. Evolutionarily conserved miRNAs in S. japonicum

Some of these conserved miRNAs have been functionally characterized in mammalian cells, C. elegans or Drosophila and were also shown to play important roles in regulating cell differentiation, cell survival, apoptosis and proliferation (Table 4). Briefly, miR-1 is a very widely conserved miRNA with specific expression in muscle and heart (Lee and Ambros, Reference Lee and Ambros2001; Lagos-Quintana et al. Reference Lagos-Quintana, Rauhut, Yalcin, Meyer, Lendeckel and Tuschl2002; Kwon et al. Reference Kwon, Han, Olson and Srivastava2005) and such diverse targets as heat shock proteins, twinfilin-1, transgelin 2, et al. (Yang et al. Reference Yang, Lin, Xiao, Lu, Luo, Li, Zhang, Xu, Bai, Wang, Chen and Wang2007; Simon et al. Reference Simon, Madison, Conery, Thompson-Peer, Soskis, Ruvkun, Kaplan and Kim2008; Yan et al. Reference Yan, Dong Xda, Chen, Wang, Lu, Wang, Qu and Tu2009; Li et al. Reference Li, Song, Zou, Wang, Kremneva, Li, Zhu, Sun, Lappalainen, Yuan, Qin and Jing2010; Nohata et al. Reference Nohata, Sone, Hanazawa, Fuse, Kikkawa, Yoshino, Chiyomaru, Kawakami, Enokida, Nakagawa, Shozu, Okamoto and Seki2011; Yoshino et al. Reference Yoshino, Chiyomaru, Enokida, Kawakami, Tatarano, Nishiyama, Nohata, Seki and Nakagawa2011) (Table 4). miR-1 targets transcription factors such as Kruppel-like factor 4 (KLF4) (Xie et al. Reference Xie, Huang, Sun, Guo, Hamblin, Ritchie, Garcia-Barrio, Zhang and Chen2011) and Paired-box transcription factor (Chen et al. Reference Chen, Tao, Li, Deng, Yan, Xiao and Wang2010c). Most interestingly, a recent study indicated that miR-1 can interact with insulin-like growth factor-1 (IGF-1) and IGF-1 receptor and this activation of the IGF-1 signal pathway reciprocally regulates miR-1 expression (Elia et al. Reference Elia, Contu, Quintavalle, Varrone, Chimenti, Russo, Cimino, De Marinis, Frustaci, Catalucci and Condorelli2009). In schistosomes, the insulin receptors have been characterized preliminarily in both S. mansoni (Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007) and S. japonicum (You et al. Reference You, Zhang, Jones, Gobert, Mulvenna, Rees, Spanevello, Blair, Duke, Brehm and McManus2010). Both species of schistosomes have two distinct insulin receptor homologues with different expression patterns. The two receptors can bind to human pro-insulin with high affinity in yeast two-hybrid assays (Khayath et al. Reference Khayath, Vicogne, Ahier, BenYounes, Konrad, Trolet, Viscogliosi, Brehm and Dissous2007; You et al. Reference You, Zhang, Jones, Gobert, Mulvenna, Rees, Spanevello, Blair, Duke, Brehm and McManus2010) and there is a link between the functions of the insulin receptor and worm glucose consumption (You et al. Reference You, Zhang, Jones, Gobert, Mulvenna, Rees, Spanevello, Blair, Duke, Brehm and McManus2010). Additionally, the IGF-1 signal transduction cascade is also subject to miRNA-145 regulation in mammalian cells (La Rocca et al. Reference La Rocca, Badin, Shi, Xu, Deangelis, Sepp-Lorenzinoi and Baserga2009). It will be interesting to investigate whether schistosome miR-1 regulates IGF-1 receptor signaling and the role it plays in parasite development, growth and host-parasite interactions. Next, miR-124 is also tissue specific being found in the nervous system of all animals studied to date (Lagos-Quintana et al. Reference Lagos-Quintana, Rauhut, Yalcin, Meyer, Lendeckel and Tuschl2002; Nelson et al. Reference Nelson, Baldwin, Kloosterman, Kauppinen, Plasterk and Mourelatos2006) and plays a vital role in neuronal development (Lim et al. Reference Lim, Lau, Garrett-Engele, Grimson, Schelter, Castle, Bartel, Linsley and Johnson2005; Krichevsky et al. Reference Krichevsky, Sonntag, Isacson and Kosik2006; Kapsimali et al. Reference Kapsimali, Kloosterman, de Bruijn, Rosa, Plasterk and Wilson2007; Makeyev et al. Reference Makeyev, Zhang, Carrasco and Maniatis2007) (Table 4). In brain tumours, miR-124 can modulate medulloblastoma cell growth by targeting cyclin dependent kinase 6 (CDK6) (Pierson et al. Reference Pierson, Hostager, Fan and Vibhakar2008) and solute carrier family 16 (SLC16A1) (Li et al. 2009). Although genome studies suggest that the schistosome nervous and sensory system may be central to parasite migration and development (Zhou et al. Reference Zhou, Zheng, Chen, Zhang, Wang, Guo, Huang, Zhang, Huang, Jin, Dou, Hasegawa, Wang, Zhang, Zhou, Tao, Cao, Li, Vinar, Brejova, Brown, Li, Miller, Blair, Zhong, Chen, Liu, Hu, Wang, Zhang, Song, Chen, Xu, Xu, Ju, Huang, Brindley, McManus, Feng, Han, Lu, Ren, Wang, Gu, Kang, Chen, Chen, Chen, Wang, Yan, Wang, Lv, Jin, Wang, Pu, Zhang, Zhang, Hu, Zhu, Wang, Yu, Wang, Yang, Ning, Beriman, Wei, Ruan, Zhao, Wang, Liu, Zhou, Wang, Lu, Zheng, Brindley, McManus, Blair, Zhang, Zhong, Wang, Han, Chen, Wang, Han and Chen2009), the molecular basis is poorly characterized. Moreover, miR-8/200b has been recognized as a critical mediator for several well characterized developmental regulatory networks such as Wnt, Notch, and TGF-beta in Drosophila or mammalian cells (Table 4). In addition, these signaling pathways have also been implicated in critical roles in schistosome development by the S. japonicum genome study (Zhou et al. Reference Zhou, Zheng, Chen, Zhang, Wang, Guo, Huang, Zhang, Huang, Jin, Dou, Hasegawa, Wang, Zhang, Zhou, Tao, Cao, Li, Vinar, Brejova, Brown, Li, Miller, Blair, Zhong, Chen, Liu, Hu, Wang, Zhang, Song, Chen, Xu, Xu, Ju, Huang, Brindley, McManus, Feng, Han, Lu, Ren, Wang, Gu, Kang, Chen, Chen, Chen, Wang, Yan, Wang, Lv, Jin, Wang, Pu, Zhang, Zhang, Hu, Zhu, Wang, Yu, Wang, Yang, Ning, Beriman, Wei, Ruan, Zhao, Wang, Liu, Zhou, Wang, Lu, Zheng, Brindley, McManus, Blair, Zhang, Zhong, Wang, Han, Chen, Wang, Han and Chen2009). Finally, miR-7 has been shown to interact with EGF (epidermal growth factor) and IGF-1 receptors in mammalian cells (Kefas et al. Reference Kefas, Godlewski, Comeau, Li, Abounader, Hawkinson, Lee, Fine, Chiocca, Lawler and Purow2008; Webster et al. Reference Webster, Giles, Price, Zhang, Mattick and Leedman2009; Jiang et al. Reference Jiang, Liu, Chen, Jin, Heidbreder, Kolokythas, Wang, Dai and Zhou2010). Both these receptor mediated signaling pathways typically promote cell survival, growth and differentiation via the activation of several integrated signaling pathways (Taguchi and White, Reference Taguchi and White2008; Hynes and MacDonald, Reference Hynes and MacDonald2009). In S. mansoni, the growth factor receptor (SER) was shown to be present predominantly in muscle tissue (Shoemaker et al. Reference Shoemaker, Ramachandran, Landa, dos Reis and Stein1992), implying a role in muscle development and function. Furthermore, the schistosome SER can bind human EGF with high affinity when expressed in heterologous vertebrate cells and this binding can lead to SER phosphorylation in S. mansoni membranes as well as activation of a conserved Ras/extracelluar regulated kinase dependent signaling pathway, suggesting the important role of EGF-receptor in the parasite development (Vicogne et al. Reference Vicogne, Cailliau, Tulasne, Browaeys, Yan, Fafeur, Vilain, Legrand, Trolet and Dissous2004; Dissous et al. Reference Dissous, Khayath, Vicogne and Capron2006; You et al. Reference You, Gobert, Jones, Zhang and McManus2011). Overall, these conserved miRNAs appear to contribute to the regulation of several signaling pathways such as IGF (miR-1/miR-7), WNT (miR-8/200b), Notch (miR-8/200b), TGF-beta (miR-8/200b), and EGF (miR-7) in other organisms and these pathways have been shown to be potentially important for regulating schistosome development.

Table 4. Summary of the targets of evolutionarily conserved miRNAs for schistosomes

POSSIBLE ROLES OF miRNAS IN SCHISTOSOME DEVELOPMENT

Putative miRNA:mRNA target pairs potentially involved in schistosome development

As described above, phylogenetically conserved miRNAs regulate a number of signal pathways such as Wnt, VEGF, and TGF-β (Kennell et al. Reference Kennell, Gerin, MacDougald and Cadigan2008; Elia et al. Reference Elia, Contu, Quintavalle, Varrone, Chimenti, Russo, Cimino, De Marinis, Frustaci, Catalucci and Condorelli2009; Webster et al. Reference Webster, Giles, Price, Zhang, Mattick and Leedman2009) in other organisms. These pathways are likely to play critical roles in schistosome development (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006; Zhou et al. Reference Zhou, Zheng, Chen, Zhang, Wang, Guo, Huang, Zhang, Huang, Jin, Dou, Hasegawa, Wang, Zhang, Zhou, Tao, Cao, Li, Vinar, Brejova, Brown, Li, Miller, Blair, Zhong, Chen, Liu, Hu, Wang, Zhang, Song, Chen, Xu, Xu, Ju, Huang, Brindley, McManus, Feng, Han, Lu, Ren, Wang, Gu, Kang, Chen, Chen, Chen, Wang, Yan, Wang, Lv, Jin, Wang, Pu, Zhang, Zhang, Hu, Zhu, Wang, Yu, Wang, Yang, Ning, Beriman, Wei, Ruan, Zhao, Wang, Liu, Zhou, Wang, Lu, Zheng, Brindley, McManus, Blair, Zhang, Zhong, Wang, Han, Chen, Wang, Han and Chen2009). Although these evolutionarily conserved miRNAs and the corresponding mRNA targets coexist in schistosomes, it remains unknown whether the miRNAs regulate similar mRNAs.

Five highly conserved miRNAs (miR-1, miR-124, miR-8/200b, miR-195, and miR-7) are predicted to interact with possible phylogenetically conserved mRNA targets in schistosomes based on analysis of miRNA:mRNA pairs predicted using RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html). Based on predicted potential targets, signaling pathways such as Wnt, VEGF and TGF-β and such important biological processes as cell cycle regulation and actin reorganization in S. japoncum may be regulated by the evolutionarily conserved miRNAs (Table 5). These predicted interactions and their roles in schistosome development suggest interesting avenues for future experimentation.

Table 5. List of some putative evolutionarily conserved miRNA:mRNA target pairs in S. japonicum

THERAPEUTIC POTENTIAL FOR SCHISTOSOMIASIS CONTROL BY TARGETING miRNAS

Given their essential roles in many biological functions, miRNAs have considerable potential anti-pathogen therapy (Wurdinger and Costa, Reference Wurdinger and Costa2007). To date, several strategies have been used to silence miRNAs in either mammalian cells or model organisms such as Drosophila and C. elegans or even in higher organisms such as mice and primate.

Firstly, antisense oligonucleotides (ASOs) complementary to the target miRNA mediate miRNA silencing. ASOs could be single strand RNA (Krutzfeldt et al. Reference Krutzfeldt, Rajewsky, Braich, Rajeev, Tuschl, Manoharan and Stoffel2005) or DNA (Esau et al. Reference Esau, Davis, Murray, Yu, Pandey, Pear, Watts, Booten, Graham, McKay, Subramaniam, Propp, Lollo, Freier, Bennett, Bhanot and Monia2006) analogues, or locked nuclear acids (LNA) (Elmen et al. Reference Elmen, Lindow, Schutz, Lawrence, Petri, Obad, Lindholm, Hedtjarn, Hansen, Berger, Gullans, Kearney, Sarnow, Straarup and Kauppinen2008) produced by chemical synthesis. The first study using ASOs for miRNA silencing was reported by Boutla et al. in 2003. In this study, the authors injected the DNA oligonucleotides complementary to 11 miRNAs into Drosophila embryos and then observed a variety of developmental defects, implying that ASO-mediated miRNA inhibition could interfere with Drosophila development. Then, chemically modified- or cholesterol-conjugated ASOs have also been used for miRNA silencing due to their relatively long stability and the high efficiency of delivery. For example, Hutvagner and co-workers demonstrated that injection of the 2′-O-methyl modified oligonucleotide complementary to the miRNA let-7 in C. elegans caused L4 larval moult defects and the inappropriate production of the larval cuticle at the adult stage (Hutvagner et al. Reference Hutvagner, Simard, Mello and Zamore2004). Then, Krützfeldt and co-workers injected chemically modified, cholesterol-conjugated single-stranded RNA analogues (termed antagomirs) complementary to miRNAs (miR-16, miR-122, miR-192 and miR-194) in mice and subsequently observed a marked reduction of corresponding miRNA levels in many organs and tissues of mice such as liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle and ovaries. Notably, the silencing of endogenous miRNAs by these antagomirs is specific, efficient and long-lasting (Krutzfeldt et al. Reference Krutzfeldt, Rajewsky, Braich, Rajeev, Tuschl, Manoharan and Stoffel2005). To move this therapeutic potential toward human applications, Elmén et al. (Reference Elmen, Lindow, Schutz, Lawrence, Petri, Obad, Lindholm, Hedtjarn, Hansen, Berger, Gullans, Kearney, Sarnow, Straarup and Kauppinen2008) designed an unconjugated, PBS-formulated locked nucleic acid-modified oligonucleotide (LNA-antimiR) and injected this LNA-antimiR (complementary to miR-122; 3 or 10 mg/kg) into a non-human primate (African green monkeys) intravenously. They observed that the injected LNA-antimiR was taken up in the cytoplasm of the hepatocytes and formed stable heteroduplexes with miR-122, leading to the depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Since mammals including humans and mice can be final hosts for schistosomes, targeting of schistosome-specific miRNAs by using these antagomirs may offer a potential for schistosomiasis treatment. Through the ASO-based gene modulation, several therapeutic molecules are in clinical trials (Goodchild, Reference Goodchild2004; Chi et al. Reference Chi, Eisenhauer, Fazli, Jones, Goldenberg, Powers, Tu and Gleave2005), but possible toxicity and off-target effects will need to be addressed (Esau, Reference Esau2008). Alternatively, morpholino oligos, which are also synthetic nucleic acid molecules, can block miRNA activity and also may eliminate off-target effects. Kloosterman and co-workers firstly employed morpholino oligos to knock down 13 miRNAs conserved in zebrafish and subsequently demonstrated that knockdown of miR-375 affected the morphology of the pancreatic islet (Kloosterman et al. Reference Kloosterman, Lagendijk, Ketting, Moulton and Plasterk2007).

Vector-based miRNA inhibition is another strategy being tested for in vivo miRNA silencing. Expression cassettes for short antisense RNAs can be engineered into lentiviral or adenoviral vectors to give stable or transient expression in cell lines or whole animals (Poy et al. Reference Poy, Eliasson, Krutzfeldt, Kuwajima, Ma, Macdonald, Pfeffer, Tuschl, Rajewsky, Rorsman and Stoffel2004; Scherr et al. Reference Scherr, Venturini, Battmer, Schaller-Schoenitz, Schaefer, Dallmann, Ganser and Eder2007). Vector-based miRNA inhibition offers several advantages compared to chemically synthesized antagomirs, including longevity, cost-effectiveness with the potential for site-specific potential by engineering suitable promoters.

miRNA inhibition can also be achieved by targeting essential factors involved in miRNA biogenesis such as Drosha, DGCR8, Dicer and Ago using small interfering RNAs (siRNAs). However, the strategy results in globally reduced mature miRNAs, leading to severe off-target effects. Additionally, Ebert et al. (Reference Ebert, Neilson and Sharp2007) reported a competitive miRNA inhibitor, termed miRNA sponges which can specifically inhibit miRNAs with a complementary heptameric seed. However, further understanding of miRNA biogenesis and the functional mechanisms is critical for the effective optimization of miRNA silencing, be it through optimal design, chemical modification and/or delivery of the miRNA antagoniser.

The use of chemically synthesized siRNAs (Cheng et al. Reference Cheng, Fu, Lin, Shi, Zhou, Jin and Cai2009) or vector-based short hairpin RNA (Zhao et al. Reference Zhao, Lei, Liu, Zhu, Ren, Wang and Shen2008) can significantly inhibit endogenous mRNAs in several different stages such as schistosomula, adult worms and sporocysts (Boyle et al. Reference Boyle, Wu, Shoemaker and Yoshino2003). Targeting of the schistosome gynaecophoral canal protein in S. japonicum and hypoxanthine-guanine phosphoribosyltransferase in S. mansoni by siRNAs has been successfully applied in vivo leading to a significant worm burden reduction (Pereira et al. Reference Pereira, Pascoal, Marchesini, Maia, Magalhaes, Zanotti-Magalhaes and Lopes-Cendes2008; Cheng et al. Reference Cheng, Fu, Lin, Shi, Zhou, Jin and Cai2009). These preliminary studies suggest in vivo therapeutic gene modulation in schistosomes by delivering small nuclear acids may have the potential for schistosomiasis treatment. Although siRNAs are not encoded in the genome of organisms unlike miRNAs which function as natural regulators of gene expression, most antagonisers for miRNA silencing are also small nuclear acids. Consequently, it is reasonable to suggest that the usage of ASOs, LNA or vector-based miRNA antisense targeting schistosome miRNAs may have the potential for schistosomiasis treatment.