关键词: 　日本血吸虫, 醛糖还原酶, RNA干扰

Abstract: 【Abstract】 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 gene 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 μg 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 culture. 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 were supplemented 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.  Conclusion  RNAi treatment based on siRNA soaking reduce the expression of SjAR gene at both mRNA and protein level.

Key words: Schistosoma japonicum, Aldose reductase, RNA interference