日本血吸虫醛糖还原酶基因RNA干扰效应的初步研究

Effect of RNA Interference Targeting Schistosoma japonicum Aldose Reductase Gene

目的研究siRNA诱导的日本血吸虫醛糖还原酶(SjAR)基因转录本和蛋白水平的RNA干扰(RNAi)效应。方法设计并合成2条针对SjAR基因的siRNA(siRNA-1和siRNA-2)和1条阴性对照siRNA(siRNA-NC)。用日本血吸虫尾蚴感染4~6周龄雌性昆明鼠(800~1 000条/鼠),13 d后灌注小鼠肝门静脉收集童虫,每(120±10)条童虫为一组,分别采用电转法和浸泡法对各组童虫进行RNAi处理。电转处理法:向电击杯中分别加入5μg siRNA-1、siRNA-2、siRNA-NC或等体积的焦碳酸二乙酯(DEPC)水(空白对照),经125 V 20 ms方波脉冲电击童虫1次后,继续培养48 h;浸泡处理法:向童虫培养基中加入siRNA-1、siRNA-2或siRNA-NC溶液至siRNA浓度为200 nmol/L,第3天时更换一半新鲜培养基并补充siRNA,共培养5 d。每处理设3个平行对照组。干扰结束后,采用TRIzol法提取虫体RNA和可溶性总蛋白,将RNA反转录为cDNA,以SjGAPDH基因为内参,实时定量PCR检测SjAR转录本相对表达水平的变化,Western blotting分析SjAR蛋白水平的变化。结果实时定量PCR结果显示,采用siRNA-1、siRNA-2和siRNA-NC电转后,虫体SjAR转录本水平分别为(90.6±16.2)%、(84.2±7.3)%和(105.9±10.3)%,与空白对照组[(100.9±16.8)%]的差异均无统计学意义(P>0.05);使用siRNA-1、siRNA-2浸泡后,SjAR转录本水平为(48.6±8.2)%和(73.4±4.7)%,均较空白对照组[(100.0±3.3%)]显著下降(P<0.01),而阴性对照siRNA-NC浸泡后的虫体SjAR转录本的表达水平为(101.43±14.33)%,与空白对照组比较表达差异无统计学意义(P>0.05)。Western blotting分析和条带定量结果显示,以空白对照组为标准,使用siRNA-1、siRNA-2和siRNA-NC浸泡后,SjAR蛋白水平分别为79.0%、97.8%和103.2%。结论使用浸泡法对日本血吸虫童虫SjAR进行RNAi处理可降低靶基因转录本和蛋白的表达水平。

Objective To investigate the effect of siRNA-induced RNA interference （RNAi） at both messenger RNA （mRNA） and protein level, targeting the Schistosoma japonicum aldose reductase （SjAR） gene. Methods Two siRNAs （siRNA-1 and siRNA-2） were designed based on SjAR geue sequence and commercially synthesized together with a negative control siRNA （siRNA-NC） showing no homology to any known S. japonicum gene sequences. Each female Kunming mice （4-6 weeks old） was infected with 800-1 000 S. japonicum cercariae and sacrificed on Day 13 post- infection for schistosomula collection. Every 120＋10 worms were selected as one group and subjected to RNAi treatment based on siRNA electroporation or soaking. In terms of the electroporation method, 5 p^g of siRNA-1, siRNA-2, siRNA-NC or equal volumes of DEPC water （blank control） were added to each cuvette separately and the schistosomula in the cuvettes subsequently underwent a square wave pulse of 125 V and 20 ms duration, followed by a 48 h euhure. As for the soaking method, solutions of the three siRNAs were added to the culture media separately to a concentration of 200 nmol/L, with equal volumes of DEPC water added as a blank control. Fresh media were changed and siRNAs weresupplemented on Day 3 amid the 5 days＇ soaking process. Three repeats were set up for each treatment. Worm RNA and total soluble proteins were extracted after the RNAi treatment by using the TRIzol method, and cDNA was synthesized by reverse transcription of the RNA. Quantitative real-time PCR （qRT-PCR） was performed next to determine the change of relative SjAR transcripts abundance after the RNAi treatment with SjGAPDH as an internal reference. Western blotting was performed to measure the expression changes of SjAR at protein level, after degradation of the SjAR transcripts by RNAi treatment. Results qRT-PCR result showed that the relative expression level of SjAR transctripts was （90.6±16.2）%, （84.2±7.3）%, and （105.9±10.3）%, respectively, following the electroporation of siRNA-1, siRNA-2 or siRNA-NC as well as the 48 h culture, none of which showed a statistically significant difference compared with the blank control group [（100.9±16.8）%]（P〉0.05）. However, after schistosomula soaked with siRNA-1 and siRNA-2 for 5 days, the levels of SjAR transcripts were reduced to （48.6±8.2）% and （73.4±4.7）%, respectively, both showing a statistically significant difference in comparison to the blank control group [（100.04±3.25）%]（P〈0.01）. The siRNA-NC treated group （negative control） exhibited no statistically significant difference compared with the blank control group（P〉 0.05）. Accordingly, as shown by Western blotting, the SjAR protein levels of worms soaked with siRNA-1, siRNA-2 or siRNA-NC were 79.0%, 97.8% and 103.2%, respectively, compared with the blank control. Conehmion RNAi treatment based on siRNA soaking reduce the expression of SjAR gene at both mRNA and protein level.