Comprehensive analysis of miRNA profiles reveals the role of Schistosoma japonicum miRNAs at different developmental stages
Schistosomiasis is an important zoonotic disease affecting up to 40 kinds of animals and 250 million people. It has been reported that the miRNAs play a role in the metabolism, differentiation, development and reproduction in many organisms. However, the roles of miRNAs regulating the development, maturation and production in schistosome in both females and males remains unclear. Here we present the dynamic transcriptome analysis of all 79 known Schistosoma japonicum miRNAs from pairing to production, including 14 days post-infection (dpi), 16, 18, 20, 22, 24, 26, 28 dpi female and male, by small RNA sequencing. The miRNA expression profiles showed time-related characteristics in male and female from paring to production, which could be clustered into three patterns, characterized by pairing stage highly expressed (cluster 1), maturating stage highly expressed (cluster 2), and egg producing stage highly expressed (cluster 3). The enrichment of miRNA cluster targeted genes in female and male were distinctly different. Network analysis of miRNAs and their target regulation showed that cluster 1 had 15 miRNAs involved in the regulation of interaction, communication, immune response in female–male and parasite–host. The other 11 miRNAs were involved in gender differentiation and the meiotic cell cycle process. In cluster 2, 11 miRNAs were involved in development and sexual maturation. In cluster 3, 45 miRNAs possibly regulate metabolism and synthesis of the substance for egg production. Analysis of the miRNA regulation network would contribute to understanding the molecular mechanism in S. japonicum development and egg production.
Schistosomiasis, a zoonotic parasitic disease, is caused by blood flukes of Schistosoma spp. Schistosomiasis has a wide range of definitive hosts, not only more than 40 kinds of animals including cattle, goats, sheep, camels, canine, swine, equine, equus ferus × asinus, macaca, felidae and rodents and avian [1, 2, 3, 4], but also affecting 258 million people in 76 countries in tropical and subtropical regions. Schistosoma japonicum (S. japonicum) is prevalent in Asia, primarily China and the Philippines [5, 6]. After infection, schistosomula migrate into the host’s bloodstream, where they develop into sexual maturation and lay eggs. Dioecy is a unique characteristic of schistosomes, in which the adult male and female live in a copula, while the female does not mature without a male [7, 8, 9]. Each paired female S. japonicum can deposit 1500–3000 eggs per day, which is about 5 to 10 times more than that of Schistosoma mansoni (S. mansoni). Thus, S. japonicum causes more severe morbidity than other schistosome species [9, 10]. Schistosome sex differentiation, maturation and egg production are the key events contributing to consequent morbidity and transmission. Understanding the molecular mechanisms of these processes is extremely urgent in current investigations on trematodes. MicroRNAs (miRNAs) are a class of small non-coding RNAs (~22 nucleotides in length), which play a crucial role in regulation of gene expression by binding to target messenger RNA (mRNA) and triggering translation repression or mRNA degradation [11, 12].
MicroRNAs have been reported to be involved in post-transcriptional regulation of gene expression during development, differentiation, proliferation, death and metabolism in a variety of organisms . To date an increasing number of schistosome miRNAs from various development stages have been identified with transcriptome analysis and deep-sequencing technique in S. japonicum [14, 15, 16, 17, 18] and S. mansoni [19, 20, 21]. miRNA and endogenous siRNA expression in S. japonicum were profiled at the stages of cercariae, lung-stage schistosomula, eggs sequestered in tissues, adults of both sexes by deep sequencing and qRT-PCR methods, which provided a broader view of small RNAs of the parasite [22, 23]. A study on sex differential expression of miRNAs in S. mansoni found that 10 of 13 microRNAs were more abundant in females than in males, exhibiting sex-biased expression patterns, and thus pointing towards a plausible involvement of miRNAs in the sex differentiation and maintenance . Sun et al. investigated the differential expression profiles of S. japonicum miRNAs in mature and immature worms, and they also analyzed miRNAs’ target genes. The results showed that more genes and metabolic pathways were regulated by miRNAs in paired mature females than in unpaired immature females . Sequencing of small RNAs from 16, 22 and 28 dpi S. japonicum showed that 18 miRNAs are expressed differently between male and female, and that by knocking down the expression of female enriched miRNAs sja-bantam and sja-miR-31, morphological alterations were observed in female ovaries . Recently, Protasio et al. reported that a novel S. mansoni miRNA family (sma-miR-277/4989) might play a dominant role in post-translational regulation in the sexual development of female schistosomes . Wang’s report  showed that S. japonicum pairing began at as early as 16 dpi. At 20 dpi, oogonium and spermatogonium were initially differentiated and mature vitelline cells firstly appeared in females. At 24 dpi, mature germ cells and vitelline glands had appeared in most of females. Meanwhile, at 20–22 dpi and 24–26 dpi, thousands of transcripts were down regulated in female worms. Therefore, 14–16 dpi, 20–24 dpi, 24–26 dpi were crucial stages for S. japonicum pairing, development and maturation . However, all known 79 S. japonicum miRNAs expression profiles and the role of miRNAs during those developmental stages (14–16 dpi, 20–24 dpi, 24–26 dpi) are currently unknown.
To gain insight into the role of S. japonicum miRNAs in the developmental process, particularly in gender differentiation and maturation, we utilized high-throughput small RNA sequencing to quantify the dynamic expression profile of miRNAs at 14, 16, 18, 20, 22, 24, 26, 28 dpi S. japonicum worms. Our results demonstrated that 79 known miRNAs had time-related characteristics in male and female. Enrichments of each miRNA cluster targets in female and male were distinctly different. Network analysis showed different miRNAs might play critical roles in the pairing, developmental and egg production stages. Our results also showed that different transferase activities were enriched in F-cluster 3, M-cluster 1 and M-cluster 3, which suggested that miRNAs might participate in the post-translational modification during different developmental stages. Global view and specific understanding of miRNA roles would contribute to study the molecular mechanism of S. japonicum developmental and maturation events.
Materials and methods
Animals, parasite and worm collection
Schistosoma japonicum was harvested by hepatic-portal perfusion from mice (C57BL/6, female, 5–6 weeks) infected with cercariae released from Oncomelania hupensis snails (Anhui isolate). The infected snails were provided by the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai. Worms were collected at 8 time points on every other day from 14 dpi through to 28 dpi. The sampled worms were washed with sterile DMEM (Dulbecco’s Modified Eagle’s medium) three times. Male and female worms were separated under light microscopy and stored in RNAlater stabilization solution (Invitrogen, USA) at −80 °C before use. Assays were processed with worms from one mouse representing one biological replicate, all assays in our study had three biological replicates.
RNA extraction, small RNA library construction and sequencing
Schistosoma japonicum total RNA was isolated using Qiagen miRNeasy Mini Kit (Valencia, CA, USA) according to the manufacturer’s instruction. The concentration and purity were evaluated spectrophotometrically using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and a NanoDrop ND2000 spectrophotometer at 260 nm and 280 nm. The small RNA library was constructed as previously described [26, 27, 28]. Briefly, 10 µg total RNA was separated by electrophoresis on a Novex 15% TBE-Urea gel (Invitrogen Co. Ltd) and RNA in length of 18–30 nt was purified and ligated to 3′ and 5′ adapters. Then for reverse transcription, we used a RT-PCR kit (Invitrogen Co. Ltd) to produce complementary DNA (cDNA). Finally, the cDNA was amplified using adapter primers, the products were purified using a 6% TBE PAGE gel (Invitrogen Co. Ltd) and sequenced with Illumina Genome Analyzer (Illumina HiSeq 2000) at the BGI (Beijing Genomics Institute, Shenzhen, China).
Analysis of sequencing data
The quality of the RNA-Seq data was first examined using the package FastQC . After a trimming process using Trimmomatic  adapters, PCR sequences and bases with low quality in the reads were clipped. A sequence read was discarded if it was below 17 bases in length. Using miRDeep v.2.0 , reads were aligned to the reference S. japonicum genome  from wormbase and known miRNAs database (downloaded from miRBase v20 ). Candidate miRNAs were identified with the miRDeep2 module using the core algorithm in RNAfold tool  to predict RNA secondary structures and evaluate the structure and signature of each putative miRNA precursor. Predicted miRNAs with significant Rand fold α level (P < 0.05) and miRDeep2 score (≥ 5) were considered as candidate miRNAs. Normalized expression profile (log scale) was generated by the quantifier module in miRDeep2 v.2.0.
Validation of miRNAs expression by qRT-PCR
RNA extraction, and reverse transcription
Total RNA of S. japonicum male or female worms were isolated using Trizol reagent (Life Technologies, USA). Genome DNA was removed from total RNA with the DNase I (Takara, Japan). For polyadenylation and reverse transcription, poly-A tails were added to total RNA (1 μg) uncontaining genome DNA with ATP by Escherichia coli (E. coli) poly (A) polymerase using miScript PCR Starter Kit (Qiagen, Germany). Controls without reverse transcriptase were included for each sample.
Real-time PCR amplification of miRNAs
We applied poly-A quantitative reverse transcription PCR (qRT-PCR) method  to amplify miRNAs. For each PCR amplification, approximately 50 ng cDNA was mixed with 10 μM forward primer and 10 μM reverse primer (universal primer) (Qiagen, Germany), 10 μL 2× QuantiTect SYBR Green qPCR master mix (Qiagen, Germany) and sterile water in a final volume of 20 μL. Cycling conditions were: 95 °C for 5 min, followed by 45 cycles of 95 °C for 15 s, 55 ± 2 °C for 30 s and 72 °C for 30 s. Dissociation curve analysis was carried out at the end of each PCR run to verify amplification specificity for each miRNA. All reactions were run in triplicate, each sample has three technical parallel replicates and each group has three biological replicates. Sj-U6 snRNA was used in the qPCR reaction as an endogenous reference with forward primer 5′-GCAAGGATGACACGCAAAT-3′ and reverse primer 5′-ATGGAACGCTTCACGAAT-3′ . Data analysis for Real-time PCR amplification efficiency and for quantifying relative miRNA expression were calculated using the 2−ΔΔCT method .
MiRNA target prediction
We used two software packages, RNAhybrid  and miRanda , to predict miRNA targets. In this study, we predicted the potential miRNA target sites not only with 3′ untranslated region (3′ UTR), but also with coding sequence (CDS) and 5′ UTR as the Ref. [11, 39, 40]. The mRNAs were based off the latest dataset of S. japonicum whole transcriptome (GenBank accession no. PRJNA343582). The cutoff was set: RNAhybrid with e-value ≤ 1e−20, minimum free energy (MFE) ≤ −20 kcal/mol. miRanda was used with a cutoff score of ≥ 140, energy cutoff ≤ −20 kcal/mol, gap opening = − 9.0 and gap extension = −4.0. Target score (i.e. the overall score of a miRNA-3′ UTRs pair in case that multiple binding sites occur) is the sum of scores for all binding sites. The miRNA potential targeted genes predicted by both tools were selected for the following network analysis.
Luciferase system reporter assay and cell culture
MiRNA mimics and NC mimics (2′-O-methyl oligonucleotides) were synthesized chemically by Genepharma (Shanghai, China), the miRNA target binding site fragments were inserted into the multiple cloning sites of pmirGLO vector dual-luciferase report system (Promega, USA). HEK293T cells were cultured in 10% fetal bovine serum in DMEM (Gibco, USA), 800 nanograms of recombinant plasmid and 20 pmol miRNA mimics (GenePharma, China) were co-transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen, USA) in 24-well plates. Firefly and renilla luciferase activity were measured after 24 h transfection by the dual luciferase assay system (Promega, USA).
MiRNA cluster and GO analysis
The miRNA clusters were processed by MeV 4.9 software based on their normalized expression level. MiRNAs expression profile in female were patterned into three clusters: F-cluster 1, F-cluster 2 and F-cluster 3. MiRNAs expression profile in male also were patterned into three clusters: M-cluster 1, M-cluster 2 and M-cluster 3.
GO IDs of target genes were obtained with the BioProject no. PRJNA343582. The Gene Ontology (GO) enrichment analysis of the 6 miRNA clusters were processed by Blast2GO  (v4.2). All potential targets from each miRNA cluster in female and male were pooled together and enrichment performed for male and female separately.
MiRNAs network analysis
Firstly, we sorted out GO terms enriched in female and male clusters, respectively. Secondly, we listed the targets enriched in each GO enrichment. Thirdly, we sorted out the miRNAs regulating those targets (from RNAhybrid and miRanda prediction) in each GO enrichment in female and male cluster. Lastly, Network was used to show the relationship between enrichment and miRNAs in cytoscape3.6.0 .
Characteristics of S. japonicum small RNAs sequencing
Dynamic profiles of S. japonicum miRNAs in female and male
The 79 known S. japonicum miRNAs clusters in female and male
sja-miR-10-5p, sja-miR-124-5p, sja-miR-2162-5p, sja-miR-2c-5p, sja-miR-2c-3p, sja-miR-2d-5p, sja-miR-3479-5p, sja-miR-3482-5p, sja-miR-3484-3p, sja-miR-3494, sja-miR-7-3p, sja-miR-190-3p, sja-miR-2a-3p, sja-miR-2e-5p, sja-miR-310, sja-miR-3483-3p, sja-miR-3498, sja-miR-3501, sja-miR-3503, sja-miR-71b-3p
sja-miR-2b-3p, sja-miR-2c-3p, sja-miR-3485-5p, sja-miR-3485-3p, sja-miR-3489, sja-miR-3491, sja-miR-3498, sja-miR-3500, sja-miR-71b-3p, sja-miR-2162-5p, sja-miR-2d-3p, sja-miR-2e-5p, sja-miR-3483-3p, sja-miR-3486-5p, sja-miR-277, sja-miR-3490, sja-miR-3492, sja-miR-3493, sja-miR-3494, sja-miR-3497, sja-miR-3499, sja-miR-3501, sja-miR-3502, sja-miR-3504, sja-miR-3506, sja-miR-36-5p, sja-miR-71b-5p, sja-miR-7-3p, sja-miR-124-5p, sja-miR-190-3p, sja-miR-2a-3p, sja-miR-2c-5p, sja-miR-2d-5p, sja-miR-310, sja-miR-3479-5p, sja-miR-3481-5p, sja-miR-3485
sja-let-7, sja-miR-219-5p, sja-miR-2a-5p, sja-miR-2e-3p, sja-miR-31-5p, sja-miR-31-3p, sja-miR-3507, sja-miR-36-5p, sja-miR-10-3p, sja-miR-124-3p, sja-miR-133, sja-miR-1, sja-miR-219-3p, sja-miR-2b-3p, sja-miR-2d-3p, sja-miR-307, sja-miR-3480-5p, sja-miR-3482-3p, sja-miR-3484-5p, sja-miR-36-3p, sja-miR-61, sja-miR-71a, sja-miR-71b-5p, sja-miR-8-5p, sja-miR-8-3p
sja-miR-124-3p, sja-miR-125a, sja-miR-190-5p, sja-miR-1, sja-miR-3488, sja-miR-3496, sja-miR-3505, sja-miR-61, sja-miR-8-3p, sja-let-7, sja-miR-10-3p, sja-miR-133, sja-miR-2162-3p, sja-miR-219-5p, sja-miR-219-3p, sja-miR-2e-3p, sja-miR-307, sja-miR-3479-3p, sja-miR-3482-3p, sja-miR-3487, sja-miR-7-5p, sja-bantam
sja-miR-125b, sja-miR-3481-5p, sja-miR-3485-3p, sja-miR-3486-5p, sja-miR-3486-3p, sja-miR-3487, sja-miR-3488, sja-miR-3489, sja-miR-3492, sja-miR-3495, sja-miR-3499, sja-miR-3506, sja-miR-7-5p, sja-bantam, sja-miR-125a, sja-miR-190-5p, sja-miR-2162-3p, sja-miR-2b-5p, sja-miR-3479-3p, sja-miR-3480-3p, sja-miR-3481-3p, sja-miR-3483-5p, sja-miR-3485-5p, sja-miR-3490, sja-miR-3491, sja-miR-3496, sja-miR-3497, sja-miR-3500, sja-miR-3502, sja-miR-3504, sja-miR-277, sja-miR-3505, sja-miR-8185, sja-miR-3493
sja-miR-10-5p, sja-miR-2a-5p, sja-miR-2b-5p, sja-miR-31-3p, sja-miR-3480-5p, sja-miR-3482-5p, sja-miR-3483-5p, sja-miR-3484-3p, sja-miR-71a, sja-miR-8185, sja-miR-125b, sja-miR-3481-3p, sja-miR-3503, sja-miR-36-3p, sja-miR-8-5p, sja-miR-3480-3p, sja-miR-3507, sja-miR-31-5p, sja-miR-3484-5p, sja-miR-3486-3p
Functional enrichment of predicted miRNA targets
Difference of miRNAs cluster targets enrichment between female and male
Specific functional enrichments
Cluster 1 (pairing stage)
Regulation of chemotaxis
Cell junction organization
Regulation of epithelial to mesenchymal transition
Cluster 2 (developmental and mature stage)
Response to BMP (bone morphogenetic protein)
Regulation of meiotic cell cycle
Cluster 3 (egg production stage)
Cluster 1 (pairing stage)
Meiotic nuclear division
Cluster 2 (developmental and mature stage)
Regulation of immune effector process
Regulation of cell growth
Regulation of response to biotic stimulus
Cluster 3 (egg production stage)
Cell communication by chemical coupling
Regulation of cholesterol homeostasis
Regulation of protein activation cascade
Network analysis between miRNAs and their targets enrichment
In order to further understand the multiple regulation roles of miRNAs during different developmental stages, we analyzed the network between miRNAs and their target enrichments in cluster 1, cluster 2 and cluster 3 (Figure 5, Additional file 10), respectively. The network showed 31 miRNAs participated in the regulation in cluster 1 (pairing stage). Among them, 20 miRNAs were specifically enriched in females, 7 miRNAs were specifically enriched in males, and 4 miRNAs were enriched both in females and males. Notably in females, 5 miRNAs regulated the leukocyte proliferation, 7 miRNAs regulated the chemotaxis, and 9 miRNAs regulated the signal transduction by protein phosphorylation. These 15 miRNAs (Figure 5, Additional file 10) might play a key role in the stimulation or attraction of female-male pairing, communication and immune response in parasite–host during pairing stage. In males, 7 miRNAs regulated sex differentiation, and 7 miRNAs regulated meiotic nuclear division. It was suggested that miRNAs may be involved in male sexual development regulation at the early paring stage . In cluster 2 (developmental and mature stage), 29 miRNAs participated the developmental and sexual maturation stage. 15 miRNAs were only enriched in females, 10 miRNAs were only enriched in males, and 4 miRNAs were enriched both in females and males. Notably, 6 miRNAs regulated meiotic cell cycle, pregnancy process in females. 5 miRNAs regulated the cell growth in males. In cluster 3 (egg production stage), 45 miRNAs participated in regulation during the egg production stage, the items of enrichments were less in female and male comparing with cluster 1 and 2. However, 34 miRNAs in female and 21 miRNAs in male were enriched in metabolic processes, which was the largest number of miRNAs observed in a process (Figure 5, Additional file 10). These miRNAs might regulate numerous material synthesis and metabolism for egg production. Notably, 2 miRNAs were enriched in chemosensory behavior, 3 miRNAs were enriched in nerve development, 3 miRNAs were enriched in body morphogenesis and 3 miRNAs were enriched in regulation of epithelial to mesenchymal transition. These 7 miRNAs in cluster 3 might play specific role during interplay in female-male and female specialized function of egg production (Figure 5, Additional file 10). Above information may provide many new clues to investigating the regulation mechanism of male and female sexual development, which deserves further attention and experimental validation.
This study focused on S. japonicum 79 known miRNAs profile (14 to 28 dpi), and the results suggested that S. japonicum miRNAs had three similar expression clusters in male and female from pairing, development, sexual maturation to egg production. The stage related miRNA clusters may regulate specific functions and play distinct roles in order to accomplish S. japonicum parasitism in host during pairing, sex-maturation and production. MiRNA is a potential factor in male–female and parasite–host interplay. Our study provided a comprehensive miRNA analysis with the goal of understanding the molecular role for miRNAs during developmental events of the schistosome life cycle.
We also looked at the target functional enrichments of “the spermatid differentiation” in females, which was interesting. It has previously been reported  that cells in the male seminal vesicle were identified as early stages of spermatogenesis, which were immature sperm, while the mature sperms were found in female seminal vesicle and oviduct. This phenomenon indicated that the spermatid maturation process might occur in female. Another possibility of this phenomenon was the sperm maturation occurred in male and the mature sperms were stored in the end of excretory bladder and then given to the female. We couldn’t observed the whole process and couldn’t improve this suppose.
Transferase activity are involved in hundreds of different biochemical pathways throughout the whole biological process, and are integral to some of life’s most important processes. Our results in transferase activity enrichments in F-cluster 3 (188, 39%), M-cluster 1 (72, 99%), M-cluster 3 (49, 100%). showed different transferase activity groups enriched in female and male. Results (Additional files 11, 12) showed 53.1% (26 of 49) of the targets were specific in M-cluster 3, 18.1% (34 of 188) targets were specific in F-cluster 3, 33.3% targets (24 of 72) were specific in M-cluster 1. Next, we also analyzed which miRNAs regulated those transferase group, results (Additional file 13) showed 10 miRNAs regulated transferase activity were specific in M-cluster 3, 7 miRNAs were specific in F-cluster 3, 12 miRNAs were specific in M-cluster 1. There were also different subcategories (Additional file 14) of transferase were enriched in F-cluster 3, M-cluster 1 and M-cluster 3. The rich transferase family member of the targets suggested that miRNA can regulate many important biological processes through post-translational modification [45, 46, 47, 48] which included of methyltransferase, acetyltransferase, glucosyltransferase, galactosyltransferase, phosphoribosyl transferase, aminotransferase, mannosyl transferase, ribosyl transferase, cytidylyl transferase, pyrophospho kinase, et al. Especially, methyltransferase and acetyltransferase were key regulatory factors in epigenetics via DNA methylation, histone methylation and histone acetylation. Okada et al.  reported that histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis, O’Carroll et al.  reported that histone H3 methyltransferase gene-Suv39h2 that displays testis-specific expression, Tachibana et al.  reported G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Disorder in histone methylation or acetylation during spermatogenesis directly affected the establishment and maintenance of epigenetic modifications, leading to spermatogenic cell abnormalities and even infertility. Even though those reports were all carried out in mammalian systems, our results indicated methyltransferase activity regulated by miRNA in schistosome might play important developmental role via post-translational modification, e.g. methylation and acetylation.
The authors declare that they have no competing interests.
WH conceived and designed this study, JY performed the analysis of data, experimental assay and drafted the manuscript. YY, HS, RZ and QL participated in the data process, MC participated in the animal experiments and miRNAs sequencing. QL and WH modified the manuscript. WH and MS coordinated preparation of the manuscript. All authors read and approved the final manuscript.
This work was funded by the National Natural Science Foundation of China (Nos. 81271867 and 31725025), International Science and Technology Cooperation Program of China (No. 2014DFA31130).
Availability of data and materials
All raw data of 48 samples small RNA sequencing were submitted to GenBank with the project accession number of PRJNA512425. Nucleotide sequences have been deposited in the Sequence Read Archive (SRA) of NCBI. The dataset supporting the conclusions of this article is included within the article and in additional files.
Ethics approval and consent to participate
All laboratory animal tests were approved by the Ethics Committee of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention in Shanghai, China (Ref. No.: 20100525-1). The use of mice in these experiments was conducted in adherence to the guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of People’s Republic of China ((2006)398).
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