| Schistosomiasis is one of the worldwide-spread parasitic diseases that causes serious healthy problems to human and other suspcetible mammals. Schistosomiasis is prevalent mainly in upland and marshland regions south of the Yangtze River in China, and our current aim about the control of schistosomiasis is to elucidate the molecular mechanism of development in schistosomes. Some research predicted the existence of the Wnt signal pathway in Schistosoma japonicum according to the sequence data of S. japonicum transcriptome and proteomics. The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway used extensively during animal development. Former study isolated the Wnt4 gene (SjWnt4) from S. japonicum, and it is the first member of the Wnt family to be identified in S. japonicum. The main function of mammal Wnt4 orhtologues is to regulate the development of the reproductive organ, whether the S. japonicum Wnt4 had similar function and the molecular mechanism of action need to be future investigated.In the present study, to elucidate the mechanism of action of S. japonicum Wnt4, its gene structure and stage-specific mRNA levels were analylzed; Anti-Wnt4 serum was prepared and used to immunohistochemical localization; RNAi was applied to explain that the Wnt4 signal protein in S. japonicum regulates downstream genes by the canonical Wnt/β-catenin pathway and its one rule is to regulate the epidermis tissue growth and oviposition; Full-length S. japonicumβ-catenin cDNA was acquired and its structural feature and stage-specific mRNA levels were also analylzed.1. The SjWnt4 structural feature and its stage-specific mRNA levelsS. japonicum genomic DNA digested by specific restriction enzymes was used as the template to generate GenomeWalkerTM libraries; Gene-specific primers were designed to amplify Wnt4 DNA fragments, and the sequence analysis between the Wnt4 DNA fragments and Wnt4 cDNA sequence showed that SjWnt4 gene is consist of 4 exons and 3 introns. As 18S rRNA the inner control, real-time quantitative PCR was performed to indicate the SjWnt4 had relative higher mRNA levels in eggs, early-stage schistosomula, 23-day male and female worms. 2. The preparation of anti-Wnt4 serum and the immunohistochemical localization of SjWnt4 proteinGene-specific primers were designed to amplify the partial SjWnt4 cDNA fragments; The PCR products and pET28a(+) vector were digested by two specific restriction enzymes and ligated together to form the pET28a-Wnt4-156 recombinant plasmid. The recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) for expression. Recombinant protein was purified and used as antigen for anti-Wnt4 serum production. Western blotting confirmed the specific binding between anti-serum and native Wnt4 protein.As anti-Wnt4 serum the primary antibody, the immunohistochemical localization pattern of the protein showed a broad distribution in the subtegumental musculature of 13 d schistosomula, 18 d schistosomula and 32 d adult worms, while Wnt4 protein was not found expressed in the testes of male worms and the ovary of female worms. It is supposed the Wnt4 protein might involved in regulating epidermis-related development.3. The pathway by which Wnt4 signal protein in S. japonicum functions elucidated by RNAiFour 19-nucleotide double-stranded short interfering RNA (siRNA) sequences of SjWnt4 were designed using online RNAi software. FAM-labeled siRNA was provided to track the entry of siRNA into the schistosomes, and fluorescence microscope was used to examine that siRNA can be introduced efficiently into 10-day-old worms; By electroporation, the most effective siRNA 708 was selected to the following tests; The effectiveness of RNAi was compared using the soaking method and electroporation in 10-day-old worms, and the results showed that both soaking and electroporation could suppress the SjWnt4 effectively while the inhibition ratio by electroporation was about twice as much as by the soaking method; Different doses were examined, and it is indicated 40μg siRNA had equal effect on inhibition of SjWnt4 expression with 50μg siRNA and meanwhile had no visible impact on the viability of the worms, so 40μg dose siRNA was used in the following studies.Based on the presumed Wnt signaling pathway of S. japonicum and the active suppression of sjWnt4 in 7-day cultured worms, six selected downstream genes involved in the three major Wnt pathways were analyzed in this study. Compared with the negative control group, the levels ofβ-catenin and GSK-3βmRNA were decreased by 45% and 39%, respectively, and these two genes are involved in the canonical Wnt/β-catenin pathway. While the mRNA levels of other four genes showed no significant differences between the control and experimental group. These results indicated that the Wnt4 signaling transduction protein functions by means of the canonical Wnt/β-catenin pathway in S. japonicum. Western blotting was used to analyze the Wnt4 andβ-catenin protein in experimental group were decreased respectively.4. The function of Wnt4 on the development of S. japonicumBy electroporation, siRNA was induced to newly-transformed schistosomula and the schistosomula were injected into Balb/c mice to finish the whole development. Six weeks later, the mice were euthanized, adult worms were examined, and the schistosome eggs was recovered from the mice livers. The results indicated that the length of worms in experimental group was shorter than control group and the liver egg burden in experimental group was less than control group. These results confirmed that one rule of Wnt4 is to regulate the epidermis tissue growth and oviposition.5. The S. japonicumβ-catenin full-length cDNA and its stage-specific mRNA levelsRACE was applied to acquire the full-length cDNA of S. japonicumβ-catenin, and the Genbank accession number was GU570442. Online software showed this molecule had the typical ARM structural feature ofβ-catenin family. Real-time quantitative PCR indicated the stage-specific mRNA levels ofβ-catenin were similar to Wnt4 in S. japonicum.In conclusion, Wnt4, the first member of the Wnt family identified in Schistosoma japonicum, regulates worm development by the canonical pathway. One of its function is to regulate the epidermis tissue growth and oviposition. Whether this protein has another functions will need further investigation and verification.
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