RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini - PubMed (original) (raw)

RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini

Jittiyawadee Sripa et al. Parasitol Int. 2011 Sep.

Abstract

Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labeled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125V in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 h after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.

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Figures

Figure 1. Opisthorchis viverrini flukes transduced by square wave electroporation with fluorescent, control Cy3-Silencer siRNA

Adult O. viverrini transfected with Cy3-siRNA using square wave electroporation, employing a single pulse for 20 ms of 125 volts in the presence of 50 μg/ml of Cy3-siRNA. The worms were observed under bright field (panel A, C, E and G) and fluorescence (panel B, D, F and H) at 0, 24 and 48 hours after electroporation; 0 hours, A, B; 24 hours, C, D; 48 hours, E, F, G, H. Panels E and G present two different worms at 48 hours after electroporation; the two different panels are included to provide a more complete representation of the signals evident in the worms at this time point. (x4 magnification, Nikon Eclipse TS100 with Epi-Fluorescence Attachment (Mercury Lamp Illuminator model name, C-SHG) (Nikon Instruments Incorporation, Japan).

Figure 2. Specific silencing of expression of the cathepsin B1 gene, _Ov_-CB-1 of Opisthorchis viverrini by dsRNA

Panel A: Real-time RT-PCR targeting _Ov_-CB-1 cathepsin B revealed >90% knockdown in specific mRNA levels when monitored at one, two and three days after introduction of dsRNAs into cultured flukes. Knockdown of _Ov_-CB-2 was also seen at days one and two. By day three, only incomplete silencing of _Ov_-CB-2 remained apparent, in contrast to the >90% knockdown of _Ov_-CB-1, indicating the silencing of _Ov_-CB-2 had commenced to wane. Panel B: In like fashion, dsRNA induced silencing of the granulin gene, Ov-grn-1 was examined. This experiment was included here as a control to examine whether other genes in addition to _Ov-_CB-1 could be silenced. It also investigated whether silencing in O. viverrini was specific since the levels of _Ov-_CB-1 and _Ov-_CB-2 were examined here as well. These latter two genes appear unrelated in sequence and function of Ov-grn-1. For both panels A and B, relative mRNA expression levels of the _Ov_-CB-1, _Ov_-CB-2 and Ov-grn-1 mRNA were calculated by comparing the dsRNA treated group to the non-treated group. The mRNA levels were normalized by comparison to actin mRNA and presented as the unit value of 2-ΔΔCt where ΔΔCt = ΔCt (treated worms) − ΔCt (non-treated worms).

Figure 3. Suppression of cathepsin B activity in Opisthorchis viverrini flukes by treatment with dsRNA specific for _Ov-_CB-1

Reduction in expression of _Ov_-CB-1 in adult O. viverrini flukes transduced by square wave electroporation with 100 μg of dsRNA targeting the _Ov-_CB-1 gene at one, two and three days after treatment (panel A). Corresponding knockdown in cathepsin B protease activity observed at one, two and three days after exposure to dsRNA. Hydrolysis of Z-Arg-Arg-AMC was monitored continuously from 0 to 300 min after addition of soluble extracts of the flukes to the substrate (panel B).

References

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RNA interference targeting cathepsin B of the carcinogenic liver fluke, Opisthorchis viverrini - PubMed (original) (raw)