Exploration Of Schistosomal Genetic Manipulation Technologies Pertaining To Small Non-coding RNAs

Schistosomiasis, caused by the blood-dwelling fluke in the genus Schistosoma, ranks as the second most serious parasitic disease after malaria throughout the world. Acute symptoms of this disease include cercarial dermatitis and a series of hypersensitivity reactions while chronic infection usually leads to lesions in urinary or hepato-intestinal systems of patients. So far there have been considerable achievements in genomic and transcriptomic studies of three major human schistosomiasis pathogens (i.e. Schistosoma mansoni, S. Japonicum, and S. haematobium) and thereby a logical next step will be the functional study aiming at both protein-encoding genes and small non-coding RNA (snc-RNA) genes.RNA interference (RNAi) is a double strand RNA (dsRNA) dependent gene silencing process that occurs in eukaryotes and viruses. SiRNA and miRNA represent two principal causative factors of RNAi. SiRNA frequently serves as a reverse genetic tool in the functional investigation of protein-encoding genes, whereas miRNA stands for endogenous gene expression modulators with pivotal physiological roles in various organisms. In the present study, we explored schistosomal genetic manipulation technologies centered of the two small RNA molecules.First of all, RNAi triggered by exogenous siRNA was applied to the functional investigation of S. japonicum aldose reductase (SjAR) gene. Previous study surmised that SjAR might participate in the antioxidant defense mechanism of worms against host attack and proved that drugs designed according to the three-dimensional structure of SjAR protein could efficiently kill worms cultured in vitro. In this section, two siRNA molecules, targeting distinct regions of SjAR transcript, were synthesized commercially in the beginning and subsequently they were separately transformed into schistosomules of S. japonicum 14 days post-infection by either soaking or electroporation method. After finishing the RNA extraction and reverse transcription steps, we adopted quantitative fluorescence PCR to test the abundance of SjAR transcripts. Corresponding result showed that compared with the negative control, expression level of SjAR transcripts in siRNA-soaked worms dropped substantially. However, the RNAi treatment based on electroporation did not induce statistically significant silencing effect on SjAR transcript. Western blotting was carried out next to test the impact of siRNA soaking on SjAR. expression at protein level and the result exhibited an obvious reduced expression of SjAR protein. No perceivable phenotypic change was observed in the siRNA treated worms under optical microscopy.Moreover, the regulative roles of endogenous miRNAs in schistosomes were studied. A new miRNA sponges technology was developed and used, in order to verify the interactions between miRNAs and their target messenger RNAs (mRNAs). Fluorescence proteins and firefly luciferase were chosen as reporter genes to denote the expression of sponge transcripts, and electroporation was utilized to express exogenous genes in schistosomal cells. Transformation of worms with any plasmids carrying either GFP or Cherry fluorescence protein gene produced no observable fluorescence in comparison to the negative control and the autofluorescence phenomenon from worms further disturbed the observation. However, when worms were transformed with firefly luciferase mRNA or plasmid DNA, their homogenate was able to catalyze consistent light emission of the substrate, which symbolized the expression of luciferase. In addition, when tandem repeats of miR-71 binding units were inserted into the downstream region of the luciferase gene, a substantial decrease of luciferase activity emerged. All of the preceding results proved the applicability of luciferase-based miRNA sponges to the functional survey of schistosomal miRNAs.At last, target genes of S. japonicum miR-71 were predicted and 5 mRNA candidates were elicited. In order to test the impact of miR-71 sponge on the expression of these 5 targets genes at protein level, their specific antibodies were needed. Therefore, E. coli system was used to obtain the 5 recombinant proteins of putative targets of S. japonicum miR-71. Finally, after the immunization step, rabbit polyclonal antibodies were successfully produced against 1 of the 5 recombinant proteins. Western blotting, in combination with miRNA sponges technology, will help us to verify the interaction between S.japonicum miR-71 and its target mRNA candidates in near future.