Analysis On Mirna Expression Of Host And Schistosomulum Among Different Suitable Rodent Animals Under Schistosoma Japonicum Infection

Schistosomiasis is caused by parasitic blood flukes, which is one of the most prevalent parasitic diseases worldwide. Nearly40kinds of mammal hosts can be infected by Schistosoma japonicum. Different kinds of host presented different suitability for S. japonicum infection. According to the growth and development of different hosts against schistosome infection, the hosts can be divided into suitable host, non-suitable host and non-permissive host. Nearly60%～70%of the adult S.japonicum can be perfused post-cercarial challenge from the suitable hosts, such as BALB/c mouse, yellow cattle, goats, and New Zealand white rabbits. Nearly10%～20%of the adult S. japonicum can be harvested from the non-suitable hosts, such as Wistar rat and water buffalo. Most of the schistosolums have incomplete development and died nearly15days post infection in Microtus fortis. Till now, M.fortis is the only non-permissive host infected with5、japonicum. Different kinds of host have unique micro-environments, which will effect the growth, development, maturation and the paring of schistosome, and eventually have different histopathological changes.MicroRNAs (miRNAs) are a class of endogenous, small noncoding RNAs that regulate gene expression at transcription. miRNA plays an important role in the control of developmental, physiological, and pathological processes, such as cellular differentiation, cell proliferation, and tumor generation. In the present study, Solexa deep sequencing was performed on the identification and analysis of the different sourced schistosomulum. Also, we identified the apoptosis pathway and the associated molecules for apoptosis of S.japonicum. The miRNA microarray was applied to analyze the miRNA expression difference between the tissues of BALB/c mouse, Wistar rat and M.fortis. Our study will provide valuable information to understanding the biology function of miRNA during Sjaponicum growth, development, host-parasite interplay, immune escape and so on. It also may be helpful for identifying the potential new drugs, biomarker and vaccine target for controlling schistosomiasis.1. Preliminary identification of miRNAs among the schistosomulum from different suitable rodent hosts and apoptosis phenomenon of S. japonicumIn our study, we applied Solexa high-throughput sequencing methods to investigate the microRNA expressed in schistosomulum from three hosts. This led us to anlayse35585878sequence reads including frequently and infrequently represented members of the RNA population. Then we have identified131miRNAs in schistosomulum from mouse,100miRNAs in schistosomulum from Wistar rat,144miRNAs in schistosomulum from M.fortis by using further bioinformatics analysis. Among the identified miRNA transcripts, there were32miRNAs common expressed in schistosomulum from the three hosts and28of them were clustered. We also identified50novel miRNAs,34were conserved miRNA that have the same seed in well-studied model organisms and16miRNA that seems to be specific to schistosome. The discovery of miRNA presented here may be helpful for better understanding the growth, development, nutrient metabolism and host-parasite interactions, which may be finally the potential new drugs targets, early diagnosis biomarker, or vaccine candidates for controlling schistosomiasis.Previously we found part of the differentially expressed miRNAs might associate with apoptosis, when analysed the miRNA of schistosomulum from different hosts. Then we focused on the signal pathway of apoptosis, in order to discovery the regulatory mechanism of miRNA and apoptosis during the growth and development of S. japonicum. Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in14days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI)(Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in14and23days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of Caspase-3and-7using the Caspase3/7Glo analysis assay. Analysis of Caspase-3and-7activity in schistosome demonstrated that both caspases were active in each developmental stage of S. japonicum, but was highest in the14days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the Caspase-3/7activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets.2. miRNA expression profile analysis of the tissues from different suitable rodent hosts after infected S.japonicum BALB/c mice were the suitable host of schistosome. In this study, an miRNA microarray was applied to investigate differences in miRNA expression in different tissues of mice before and10days post infection. In total,220miRNAs were detected in different tissues of the BALB/c mice before and after infection, including8miRNAs in liver,8in spleen and28in the lungs with up-regulated expression, and3miRNAs in liver,5in spleen and23in the lungs with down-regulated expression in mice10days post infection with schistosoma. The functions of these differentially expressed miRNAs are related mainly to the immune response, nutrient metabolism, cell differentiation, apoptosis, and signal pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the differentially expressed miRNAs revealed that many important biological pathways are triggered by schistosome infection in BALB/c mice, such as the MAPK signaling pathway, insulin signaling pathway, Toll-like receptor signaling pathway and TGF-β signaling pathway. The results indicated that miRNAs may be an important regulator of schistosome-host interaction in the early phase of S. japonicum infection. It also have an effect on the growth an development of S. japonicum in different hosts.When compared to the murine permissive host of Schistosoma japonicum, Wistar rats are less suitable to S. japonicum infection, and are considered to provide a less suitable microenvironment for parasite growth and development. To investigate the regulatory mechanisms provided by miRNA in the schistosome-infected rat model, we utilized a miRNA microarray to compare the progression of miRNA expression within different tissues before and10days after schistosome infection. Among the analyised miRNAs,16within the liver,61within the spleen and10within the lung, were differentially expressed in infected Wistar rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways are triggered after infection with S. japonicum in Wistar rats. These include the signal transduction mechanisms associated with the Wnt and MAPK signaling pathways, cellular differentiation, with a particular emphasis on adipocyte and erythroid differentiation. The results presented here include the identification of specific differentially expressed miRNAs within the liver, lungs and spleen of Wistar rats. These results highlighted the function of host miRNA regulation during an active schistosome infection.The reed vole M. fortis is the only mammal known in China in which the growth, development and maturation of S.japonicum is prevented. It might be that the anti-schistosomiasis mechanisms of M. fortis associate with microRNA-mediated gene expression, given that the latter has been found to be involved in gene regulation in eukaryotes. In the present study, the difference between pathological changes in tissues of M. fortis and of mice Mus musculus post-schistosome infection were observed by using hematoxylin-eosin staining. In addition, microarray techniques were applied to identify differentially expressed miRNAs in the same tissues before and post-infection to analyze the potential roles of miRNAs in schistosome infection in these two different types of host. Histological analyses showed that S. japonicum infection in M. fortis resulted in a more intensive inflammatory response and pathological change than in mice. The microarray analysis revealed that388miRNAs were expressed in both species, with11in liver,25in spleen and28in lung being differentially expressed in M. fortis. Further analysis revealed that important signaling pathways were triggered after infection by S. japonicum in M. fortis but not in the mice. These results provide new insights into the general mechanisms of regulation in the non-permissive schistosome host M. fortis that exploits potential miRNA regulatory networks, such as inflammatorry reaction, immune regulation, nutrition and metabolism. The differentially expressed miRNAs might related with the anti-schistosomiasis of M. fortis.