Effects Of Inhibition On Schistosoma Japonicum Wnts Secretion Associated Proteins And Characterization Of S.Japonicum Wnt1

Studies on Schistosome infecting human and various mammals have great significance on public health. Wnt signaling pathway is a conserved signaling pathway which involved in some important biological processes like embryonic development and growth, adult homeostasis. Because the secretion of Wnt proteins is the key point for the activation of the Wnt signaling pathway, in this project, Wnts secretion assosicated proteins were blocked by inhibitors or RNAi, and effects of inhibition on Schistosoma japonicum(S.japonicum) Wnt signaling pathway were evaluated, to increase the understanding the effects of the Wnt signaling pathway on the growth, development and homeostasis of S. japonicum. Meanwhile, SjWnt1 gene was identified and characterized.1.The study of the inhibition on SjWnt secretion-associated proteinsSchistosomes in vitro and in mices were administrated by C59,an effective inhibitor of Porcupine(Porcn)which is an important protein in Wnts secretion event. By comparing the mRNA levels of different members in Wnt signaling pathway through qPCR, we comfirmed that 20 nmol/L was the optimal concentration for C59. Six days after the administration of C59, Wnt signaling pathway of 14-day-old schistosomulum in vitro began to be inhibited effectively and persistently; Seven days after the administration of C59, the inhibition of Wnt signaling pathway of both 14-day-old and 7-day-old schistosomulum in vitro was the the most significant; The inhibition effect on 14-day-old Schistosomulum by C59 was more effective than 7-day-old schistosomulum. The qPCR results showed that mRNA of Pro-apoptotic factors and DNA damage-related factors were upregulated in 7-day-old and 14-day-old Schistosomulum administrated by C59, and upregulation degree in 14-day-old Schistosomulum were greater than 7-day-old, indicated that the inhibition of Wnt signaling pathway could result in the instability in internal environment, leading to the upregulation of the level of apoptosis. The more effective inhibition on Wnt signaling pathway, the more cells trend to apoptosis. We observed the surface structure and the ultrastructure of 14-day-old S.japonicum, whose Wnt signaling pathway were inhibited. The results showed that while the surface structure of worms in C59 group had no significant differences, suggesting no direct stimuli and lesions of C59 to body barrier of the worm, the ultrastructure of them became obscure and their intestine epitheliums fell off and intestine microvills were partly broken, compared to those in DMSO group and blank group. And these changes were consistent with the result of up-regulated apoptotic-related factors in C59 group. When S.japonicum in mices were administrated by C59, mRNA of the corresponding genes in downstream of Wnt/β-catenin, Wnt/PCP and Wnt/Ca2+ were also down-regulated and ones of Wnt/β-catenin and Wnt/Ca2+ were greater down-regulated, indicated that the Wnt/β-catenin and Wnt/Ca2+ were more active in Schistosomula stage. C59 can be used to the inhibition experiments of Wnt signaling pathway of S.japonicum in mice.The ORF sequence of the Porcn gene of S.japonicum(SjPorcn) was obtained by Race technique; Three amino acids were highly conserved in SjPorcn protein. When SjPorcn gene was knocked down by RNAi, mRNA of the corresponding genes in downstream of Wnt/β-catenin, Wnt/PCP and Wnt/Ca2+ were also down-regulated in different degrees, which is similar to the effect of C59.We isolated and identified the Wls gene of S.japonicum(SjWls). Sequence analysis indicated that SjWls contained a conserved lysine residue. The amino acid sequence “KEAFE” at the carboxyl terminus of SjWls, which is critical for subcellular localization of Wls. The tissue localization analysis of SjWls protein showed that the protein is widely distributed in acetabulum, gynecophoral canal, reproductive organs and other tissues of S.japonicum, suggesting that Wnt proteins are distributed in various tissues. After SjWls gene was knocked down, mRNA of the corresponding genes in downstream of Wnt/β-catenin, Wnt/PCP and Wnt/Ca2+ were also down-regulated.2. Characterization and expression profile analysis of Wnt1 in different stages of S.japonicumWe isolated and identified the Wnt1 gene of S.japonicum(SjWnt1). Sequence analysis indicated that SjWnt1 contained the conservative domain of Wnt protein family, but lacked one conservative cysteins than other Wnt1 in higher organisms. Relative quantification result presented that SjWnt1 expressed in all development stages of S.japonicum, and expression level of SjWnt1 in egg and 7-day-old schistosomulum was higher than other development stages, suggested that Wnt1 signaling pathway had an important role on egg development and Schistosomulum cell differentiation. We also found that SjWnt1 mRNA level in maldevelopment worm was higher than normal worm, which suggested that Wnt pathway was retarded in maldevelopment worm. When SjWnt1 gene was knocked down, mRNA of the corresponding genes in downstream of Wnt/β-catenin, Wnt/PCP and Wnt/Ca2+ were also down-regulated, indicated that SjWnt1 may might active the Wnt pathway by these three transduction pathway.In this study, through the inhibition study on Wnt secretion associated proteins of S.japonicum, the optimal concentration and time-effect of C59, the specific inhibitor of Porcn, was determined, and its inhibition effect on schistosomulum in vivo or in vitro was analyzed. Effective siRNAs of Porcn and Wls, which are Wnts secretion associated genes, were screened, and effect of inhibition on Wnt signaling pathway after Porcn and Wls down-regulated were estimated. Above research, established a primary experiment method to inhibit the Wnts family, and help to understand this pathway regulation mechanism on schistosome growth or development. Meanwhile, SjWnt1 was identified and characterized, increase the understanding of the function of SjWnt1 on Schistosome development.