| Nudix hydrolase is one kind of hydrolysis enzymes. It can hydrolyze various organic phosphates, including nucleoside diphosphate, nucleoside triphosphate, dinucleotide phosphate, diphosphoinositide polyphosphate, nucleotide sugar and RNA caps. By previous work, We conjecture that Trichinella spiralis Nudix hydrolase(Ts Nd) may be associated with Trichinella spiralis muscle larva(ML) invade the host small intestinal mucosa.The aim of this study was to investigate the functions of T. spiralis Nudix hydrolase(Ts Nd) during the larval invasion of intestinal epithelial cells(IECs), development and survival in host by RNAi. The Ts Nd-specific double-stranded RNA(ds RNA) and small interference RNA(si RNA) were designed to silence the expression of Ts Nd in T. spiralis larvae. Ds RNA and si RNA were delivered to the larvae by soaking incubation or electroporation. Silencing effect of Ts Nd transcription and expression was determined by real-time PCR and Western blotting, respectively. The infectivity of larvae treated with ds RNA was investigated by the in vitro larval invasion of IECs and experimental infection in mice. At the same time, design a ds RNA that homologous with Trichinella spiralis proteasome beta 7 subunits(Tspst) to verify the gene specificity of RNA interference. Materials and Methods1. Parasites and experimental animalsThe isolate(ISS534) of T. spiralis used in this study was obtained from domestic pigs in Nanyang city of Henan Province, China. The Trichinella isolate was maintained by serial passage in Kunming mice every 6-8 months. Speci?c pathogen-free(SPF) male BALB/c mice aged 5 weeks were purchased from the Experimental Animal Center of Henan Province. All experimental procedures were approved by the Life Science Ethics Committee of Zhengzhou University and were consistent with the NIH Guidelines for the Care and Use of Laboratory.2. Recombinant plasmid and immune serumThe recombinant plasmid p GEM-T-Ts Nd containing T. spiralis Nudix hydrolase gene(Ts Nd) and p MAL-C2X-Tspst containing Trichinella spiralis proteasome 7 beta subunit gene(Tspst) were constructed and maintained by our laboratory. The r Ts Nd or r Tspst protein was expressed in E. coli under 0.5 m M IPTG induction as described previously. The mouse immune sera against r Ts Nd or r Tspst were as collected from the immunized mice at one week after the last immunization.3. Design and synthesis of ds RNA and si RNALog in Genebank to query m RNA sequence of trichinella spiralis Nudix hydrolase and trichinella spiralis proteasome beta 7 subunits m RNA sequence.(1) The design and synthesis of ds RNA. According to the selection principles of ds RNA target sequence. Selection of trichinella spiralis Nudix hydrolase m RNA650 ~ 1301 bp and proteasome beta 7 subunits m RNA88 ~ 742 bp as two ds RNA target sequence. According to the target sequence design PCR primers containing T7 promoter. To as the template for PCR amplification of PCR products containing T7 has promoter。recombinant plasmid containing purpose gene was used as the template of PCR amplification, the PCR products containing T7 promoter could used as ds RNA in vitro transcription templates. Then in vitro transcription for ds RNA- Nudix and ds RNA- beta 7.(2) The design and synthesis of si RNA. According to the trichinella spiralis Nudix hydrolase gene m RNA sequence and the principle of si RNA design, Using si RNA si Direct online design software version 2.0(http://sidirect2.rnai.jp) design three si RNA and one NC si RNA(Negative control si RNA). Then sending si RNA sequences to Invitrogen company for synthesis. The same NC si RNA labeled with FAM was used to monitor the transfection efficiency.3 Si RNA or ds RN A Delivery to Trichinella spiralis muscles larvaeT. spiralis ML were recovered from the infected mice at 42 days post infection(dpi) by artificial digestion as described previously, and washed three times in 0.9% saline solution. Ds RNA, si RNA, control si RNA and FAM labbled control si RNA were delivered to the larvae by soaking incubation or electroporation. Muscle larvae treated with FAM labbled control si RNA can be observed fluorescent signal under fluorescence microscope, to determine the success of si RNA into the Trichinella spiralis muscles larvae.4 Detection of RNA interference effectTranscription and expression level of Ts Nd in larvae treated with ds RNA and si RNA was determined by real-time PCR and Western blotting, respectively.5 Ability of ds RNA- or si RNA-treated larvae to invade IECs in vitroTo observe the effect of RNAi on the ability of larvae to invade IECs, the IECs were grown to confluence in 24-well plates. Each cell monolayer was overlaid with approximately 100 larvae treated with ds RNA or untreated at 18 h after soaking or electroporation, suspended in 0.5 ml of semisolid medium(serum-free DMEM containing 15 m M HEPES and 1.75% agarose). The 24-well plate was incubated at 37°C in 5% CO2 for 2 h. The larval invasion of IECs was observed using inverted phase-contrast microscope(Olympus, Japan), and the number of larvae in the cell monolayer was counted.The invasion percentage of ds RNA treated larvae were compared with those of untreated group..6. Development and survival of si RNA-treated larvae in miceTo examine the infectivity of ds RNA-treated larvae, BALB/c mice were divided into 5 groups of 20 animals each. Each group was orally inoculated with 300 T. spiralis larvae electroporated with ds RNA-Ts Nd, larvae soaked with ds RNA-Ts Nd, lipefactin(lip) 2000 or PBS. Ten mice from each group were sacrificed at 6 dpi, and the AW were collected from the intestine of infected mice and counted. The fecundity of recovered female worms was observed after being incubated individually in each well of 24-well plate with 1640 medium at 37°C in 5% CO2 for 72 h, and the number of NBL produced by each female worm was counted. The muscle larvae were collected from the remaining 10 mice of each group at 35 dpi by artificial digestion as described previously. The worm reduction was calculated based on the mean number of AW or ML collected from the group treated with ds RNA-Ts Nd compared with the untreated group.Results1 AGE(agarose gel electrophoresis) show that the synthesis of ds RNA-Ts Nd and ds RNA-β7 is very successful.2 fluorescence signal show that FAM NC si RNA was imported into Trichinella spiralis muscles larvae successfully.3 By the result of Real-time PCR, we known that both ds RNA-Ts Nd and si RNA-275 could interfer the expression of Trichinella spiralis Nudix hydrolase m RNA. 1d post treated by ds RNA-Ts Nd, compared with control, the expression of Trichinella spiralis Nudix hydrolase m RNA reduced 61.0%(P < 0.05)4 The result of Western blotting showed that both ds RNA-Ts Nd and si RNA-275 could interfer the expression of Trichinella spiralis Nudix hydrolase. 3d and 4d post treated by ds RNA-Ts Nd, compared with control, the expression of Trichinella spiralis Nudix hydrolase reduced a lot. Compared with si RNA-425 and si RNA-715, si RNA-275 could cause more obvious RNA interference.5 The result of Real-time PCR showed, compared with control, the expression of Trichinella spiralis Nudix hydrolase m RNA in the muscles larvae treated by ds RNA-Ts Nd reduced 74.6%(P<0.05), but the expression of proteasome 7 beta subunit(β7) m RNA did not reduce obviously(P > 0.05). The expression of Trichinella spiralis Nudix hydrolase m RNA in the muscles larvae treated by ds RNA-β7 did not reduce obviously(P>0.05), but the expression of proteasome 7 beta subunit(β7) m RNA reduced 73.2%(P<0.05).6. ds RNA-mediated silencing of Ts Nd inhibits the larval invasion of IECsKnockdown of Ts Nd m RNA by RNAi inhibited the T. spiralis larval invasion of IECs in vitro. After soaking for 18 h, the invasion rate of the larvae soaked with 20, 30, ds RNA-Ts Nd displayed a 83.4% reduction in adult worms and 69.5% reduction in muscle larval burden compared with the mice inoculated with untreated larvae, and these differences were statistically significant(P<0.05). The difference in adult and larval burden in mice inoculated with the larvae soaked or electroporated with ds RNA-Ts Nd was statistically significant(χ2adult =24.442, χ2larvae =17.419, P<0.05), demonstrating that electroporation had a higher efficiency than soaking in inhibiting the larval development and survival in mice.After the larvae were eletroporated with 20, 30, 40, 50 and 60 ng/μl ds RNA-Ts Nd for 18 h, the larval invasion rate was 52.6%, 43.8%, 39.5%, 33.8%, and 30.8%, respectively; whereas the invasion rate of the untreated was 58.3%(Fig. 5). Except for the larvae treated with 20ng/μ l ds RNA-Ts Nd, the differences of invasion rate between ds RNA-Ts Nd treated groups and untreated group were statistically significant((χ230 =4.258, χ240 =7.044, χ250 =12.956, χ260 =16.099; P < 0.05), indicating the reduction in larval invasion of IECs was ds RNA-dose dependent(r =-0.98707). The difference in inhibiting the invasion of IECs by the larvae soaked or electroporated with ds RNA-Ts Nd was not statistically significant(P>0.05).The invasion rate of the larvae eletroporated with 1, 1.5, 2, 2.5 or 3μM si RNA-275 for 18 hours was 50.3%, 42.6%, 37.0%, 33.6%, and 30.6%, respectively; while the invasion rate of the larvae treated with control si RNA and untreated group 59.4% and 58.3%, respectively. Except for the larvae treated with 1μM si RNA-275, the differences of invasion rate between si RNA-275 treated groups and control si RNA-treated group were statistically significant(c21.5 = 5.873, χ22 = 5.873, c22.5 = 5.873, c23 = 5.873; P<0.05), indicating the reduction in larval invasion of IECs was si RNA-dose dependent(r =-0.97941).7 Mice inoculated with T. spiralis larvae transfected with ds RNA-Ts Nd by soaking displayed a 49.9% reduction in adult worms and 39.9% reduction in muscle larval burden compared with the mice inoculated with untreated larvae, and these differences were statistically significant(P < 0.05). There was no significant reduction of adult worm burden and muscle larval burden in mice inoculated with larvae soaked with lip2000 compared to mice inoculated with untreated larvae(Fig. 6). For electroporation, mice inoculated with T. spiralis larvae electroporated withMice inoculated with larvae electroporated with si RNA1743 displayed a 63.6% reduction in intestinal adult worms and 68.8% reduction in muscle larval burden compared with the mice inoculated with control si RNA-treated larvae(P<0.05). There was no significant reduction of adult worms and muscle larvae in mice inoculated with larvae electroporated with control si RNA compared to mice inoculated with untreated larvae(P>0.05)(Fig. 5). The in vitro newborn larval production in 72 h of the female adult collected from mice inoculated with the larvae treated with si RNA-275, control si RNA and PBS was 70.70±3.48, 74.06±6.64 and 72.85±4.69, respectively(P>0.05). Conclusions1 Successfully synthesized ds RNA-Ts Nd and ds RNA-β7 by in vitro transcription.2 Both ds RNA-Ts Nd and si RNA can cause silence of Ts Nd.3 Ds RNA- mediated RNAi is gene specific.4 RNAi-mediated silencing of Ts Nd inhibits the larval invasion of IECs5 Infection ability of Trichinella spiralis muscle larva silented by RNAi is reduced.6 When the Trichinella spiralis muscle larva silented by RNAi develope into female worms, there is no changes of the female fecundity. 40, 50 and 60 ng/μl ds RNA-Ts Nd was 54.4%, 44.7%, 39.2%, 35.3%, and 32.7%, respectively; while the invasion rate of the larvae treated with lip2000 or untreated larvae was 61.7% and 63.1%(Fig. 5). Except for the larvae treated with 20ng/μL ds RNA-Ts Nd, the differences of invasion rate between ds RNA-Ts Nd treated groups and untreated group were statistically significant(χ230 = 6.640, χ240 = 10.982, χ250 = 16.778, χ260 = 19.317; P<0.05), indicating the reduction in larval invasion of IECs was ds RNA-dose dependent(r =-0.96798). |
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