Parasitol Res DOI 10.1007/s00436-015-4468-1

ORIGINAL PAPER

Comparative characterization of microRNAs in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice Hongxiao Han 1,4 & Jinbiao Peng 2 & Yang Hong 1 & Zhiqiang Fu 1 & Ke Lu 1 & Hao Li 1 & Chuangang Zhu 1 & Qiuhua Zhao 4 & Jiaojiao Lin 1,3

Received: 14 February 2015 / Accepted: 3 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967–76, 2010). Compared with permissive BALB/c mice, rats are less susceptible to S. japonicum infection and are considered to provide an unsuitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), via the regulation of gene expression at the transcriptional and post-transcriptional levels, may be responsible for developmental differences between schistosomula in these two rodent hosts. Solexa deepsequencing technology was used to identify differentially expressed miRNAs from schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection. The deepsequencing analysis revealed that nearly 40 % of raw reads

(10.37 and 10.84 million reads in schistosomula isolated from Wistar rats and BALB/c mice, respectively) can be mapped to selected mirs in miRBase or in species-specific genomes. Further analysis revealed that several miRNAs were differentially expressed in schistosomula isolated from these two rodents; 18 were downregulated (by 2-fold) (expression levels in rats compare with those in mice). Additionally, three novel miRNAs were primarily predicted and identified. Among the 41 differentially expressed miRNAs, 4 miRNAs had been identified with specific functions in schistosome development or host-parasite interaction, such as sexual maturation (sja-miR-1, sja-miR-75p), embryo development (sja-miR-36-3p) in schistosome, and pathogenesis of schistosomiasis (sja-bantam). Then, the

Hongxiao Han and Jinbiao Peng contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00436-015-4468-1) contains supplementary material, which is available to authorized users. * Jiaojiao Lin [email protected]

Chuangang Zhu [email protected] Qiuhua Zhao [email protected]

Hongxiao Han [email protected] Jinbiao Peng [email protected]

1

Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, 518 Ziyue Road, Minhang Shanghai 200241, China

Zhiqiang Fu [email protected]

2

Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China

Ke Lu [email protected]

3

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

4

Minhang Animal Disease Control Center, Shanghai 201109, China

Yang Hong [email protected]

Hao Li [email protected]

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target genes were mapped, filtered, and correlated with a set of genes that were differentially expressed genes in schistosomula isolated from mice and rats, which we identified in a S. japonicum oligonucleotide microarray analysis in a previous study. Gene Ontology (GO) analysis of the predicted target genes of 13 differentially expressed miRNAs revealed that they were involved in some important biological pathways, such as metabolic processes, the regulation of protein catabolic processes, catalytic activity, oxidoreductase activity, and hydrolase activity. The study presented here includes the first identification of differentially expressed miRNAs between schistosomula in mice or rats. Therefore, we hypothesized that the differentially expressed miRNAs may affect the development, growth, and maturation of the schistosome in its life cycle. Our analysis suggested that some differentially expressed miRNAs may impact the survival and development of the parasite within a host. This study increases our understanding of schistosome development and host-parasite interactions. Keywords MicroRNA (miRNA) . Schistosoma japonicum (S. japonicum) . Wistar rats

Background Schistosomiasis is one of the most serious zoonotic diseases caused by dioecious flatworms in the genus Schistosoma. Nearly 200,000 people are currently infected with Schistosoma japonicum in China, and another 60 million are at risk of infection (Zhou et al. 2011). The control of schistosomiasis relies mainly on praziquantel, a pyrazinoisoquinoline derivative that is effective against all clinically relevant species of schistosomes (Xu et al. 2014). However, the extensive use of this drug has led to serious concerns about the future development of drug resistance, as well as the potential for frequent reinfections of humans and domestic animals in endemic areas (Han et al. 2013a). It is reported that more than 40 species of mammals, including cattle, sheep, goats, rabbits, mice, etc., are naturally infected by S. japonicum in China. Mice (Mus musculus) are permissive hosts of S. japonicum, and they support the full growth, development, and sexual maturation of the parasite. In contrast, rats (Rattus norvegicus) are less susceptible or semipermissive because they do not provide a suitable microenvironment that is conducive for parasite growth and development (Han et al. 2013b; Silva-Leitao et al. 2009). The life cycle of S. japonicum in rat hosts is unsustainable due to the low survival rate, compared with that of mice, of cercariae that penetrate through the skin, and much fewer schistosomula successfully migrate from the liver portal circulation into the mesenteric veins. Additionally, in rats, adult female worms

have a lower egg-laying rate, and they lay increased numbers of immature eggs (Han et al. 2013b; Peng et al. 2011b). MicroRNAs (miRNAs) are a class of endogenous, small noncoding RNAs (18–25 nucleotides in length) that are generated from endogenous transcripts and form hairpins. miRNAs regulate gene expression indirectly at the transcriptional level via the indirect regulation of transcription factor expression (Carthew and Sontheimer 2009; Kim 2005). Additionally, miRNAs regulate gene expression at the posttranscriptional level by binding to the 3′ untranslated regions (UTRs) of target cellular mRNAs, thereby inhibiting translation and inducing mRNA degradation (Guo et al. 2010). miRNAs play fundamental roles in diverse biological and pathological processes, including growth, metabolism, development, and cell differentiation, as well as by their ability to respond to environmental and developmental signals (Ambros 2003; Hoefig and Heissmeyer 2008; Sayed and Abdellatif 2011). Recently, some miRNAs involved in schistosome sexual maturation and development have been identified, which provides insight into the potential role of miRNAs in schistosome development, growth, maturation, and host-parasite interactions (Cheng et al. 2013; de Souza Gomes et al. 2011; Zhu et al. 2014). In the present study, we used the deep-sequencing technique to identify differentially expressed miRNAs in schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection, and further predicted its potential function. The results provide useful comparative information to better define the function of miRNAs involved in the development, growth, and maturation of the schistosome life cycle. This information will also enable the identification of the parasite molecular mechanisms associated with the growth retardation of schistosomula in Wistar rats.

Materials and methods Host animals and parasites All animal care and experimental procedures were conducted according to the guidelines for animal use in toxicology. The study protocol was approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS). BALB/c mice (8 weeks, male, 20 g) and Wistar rats (8 weeks, male, 150 g) were purchased from the Xipu’erbikai Experimental Animal Co., Ltd. (Shanghai, China) and Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China), respectively. S. japonicum (Chinese mainland strain, Anhui isolate) was maintained in New Zealand rabbits and Oncomelania hupensis snails at the Shanghai Veterinary Research Institute, CAAS. Wistar rats and BALB/c mice were infected

Parasitol Res

with 2000 and 200 cercariae, respectively. Infected animals were perfused with 37 °C pre-warmed phosphate-buffered saline (PBS) at 10 days post-infection, and the schistosomula were collected. Special attention was paid to remove all host tissue from the isolated parasites through multiple washings with PBS (pH 7.4) at 37 °C. Total RNA isolation, small RNA library construction, and sequencing Small RNA was isolated from schistosomula using the Ambion mirVana miRNA Isolation Kit, according to manufacturer’s instructions (Life Technologies, Grand Island, NY, USA). The quality of RNA was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C. Approximately 20 μg of total RNA was used for library preparation and sequencing. The RNA was converted to single-stranded complementary DNA (cDNA) using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), and subsequently amplified in 15 cycles using a small RNA primer set (Illumina, San Diego, CA, USA) and Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA). The PCR products were size fractionated and recovered for sequencing using a Genome Analyzer IIe (Illumina) according to manufacturer’s instructions.

MFOLD software (Version 2.38, http://mfold.rna.albany. edu/?q=mfold/RNA-Folding-Form). Predicted gene targets of differentially expressed miRNAs and bioinformatics analysis Targets of the miRNAs were predicted using the online softw a r e Ta r g e t S c a n S ( h t t p : / / g e n e s . m i t . e d u / t s c a n / targetscanS2005.html) in conjunction with miRanda (http:// www.microrna.org/microrna), PicTar (http://pictar.org/), and RNAhybrid (http://140.109.42.4/cgi-bin/RNAhybrid/ RNAhybrid.cgi)(Huang da et al. 2009). The target genes were mapped, filtered, and correlated with differentially expressed genes in mice and rats using a S. japonicum oligonucleotide microarray from our previous research (GEO accession no. GSE25728)(Peng et al. 2011a). The target genes were analyzed in terms of their Gene Ontology (GO) categories using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene annotation tool (http://david.abcc.ncifcrf.gov/)(Huang da et al. 2009). Validation of miRNA expression with quantitative PCR (qPCR) analysis Differentially expressed miRNAs were examined using qPCR with SYBR green (Xu et al. 2013). U6 RNA was selected as a housekeeping miRNA to normalize the miRNA expression

Data processing and bioinformatics analysis After masking the adaptor sequences and removing contaminated reads, clean reads were processed using Genome Analyzer Pipeline software (Illumina). Clean reads were then subjected to a series of data filtration steps, using the statistics of mammalian miRNAs in miRBase 20.0. Mappable sequences were obtained using the ACGT101-miR program. Reads that matched rRNAs, tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), repeat sequences, and other noncoding RNAs (ncRNAs) deposited in Rfam 8.0 or NCBI GenBank (http://www.ncbi.nlm.nih.gov/ GenBank) were excluded. The retained 18–26 nt reads in each library were mapped onto the draft S. japonicum genome sequence (ftp://down:[email protected]:2121/ subjectData/schistosoma/) and (ftp://ftp.sanger.ac.uk/pub/ pathogens/Schistosoma/mansoni/genome/smav5.2.chr.fa) using the Short Oligonucleotide Alignment Program (SOAP) (http://soap.genomics.org.cn). Then, we aligned the predicted microRNAs to all known deuterostomal mature miRNA sequences in miRBase version 20.0 (http://microrna.sanger. ac.uk) to determine their novelty. Other small RNAs were further analyzed to predict new miRNAs using MIREAP (http://sourceforge.net/projects/mireap). Secondary structure prediction of individual miRNAs was performed using

A Raw reads of schistosomula from Wistar rats 3% 4% 0.2% Number of reads removed due to 3ADT not found: 344,398 (3.3%) Number of reads removed due to

c mice.

More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967...
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