Abstract:

Objective To investigate the effect of siRNA interference with EgRad9 gene expression on DNA oxidative damage in protoscoleces of Echinococcus granulosus. Methods EgRad9-siRNA and scrambled iRNA were transfected into protoscoleces of Echinococcus granulosus by electroporation. It included EgRad9-siRNA-354, -614, and -971 interference groups, scrambled control and blank control groups. Three days after electroporation, the protoscoleces were treated with the maximum tolerated concentration of H₂O₂ for 1 h, stained by eosin, and observed under an inverted fluorescence microscope to calculate the survival rate of protoscoleces. The cultural medium in each group was collected for alkaline phosphatase activity assessment. RNA was extracted from protoscoleces using TRIzol, and EgRad9 mRNA expression was assessed by qRT-PCR. The single cell gel electrophoresis (comet assay) was used to detect DNA damage before and after transfection. The content of reactive oxygen species (ROS) in protoscoleces was assessed using an ROS detection kit. Data were analyzed using the SPSS 22.0 software. Results The maximum tolerated concentration of H₂O₂ for protoscoleces of Echinococcus granulosus was 0.4 mmol/L, and the intervention duration was 1 h. The siRNA sequences were effectively transfected by electroporation. Green fluorescent spots were clearly seen in the interference groups and scrambled control group, while none appeared in the blank control group. The survival rates of protoscoleces in the EgRad9-siRNA-354, -614, and -971 interference groups were (42.99 ± 4.03)%, (29.86 ± 5.87)% and (56.48 ± 4.64)%, respectively. Thus the EgRad9-siRNA-614 was considered as the most effective interfering sequence. The alkaline phosphatase activity in the EgRad9-siRNA-354, -614, and -971 interference groups was 0.024 ± 0.001, 0.004 ± 0.001, and 0.039 ± 0.002, respectively, with significant differences between EgRad9-siRNA-614 versus scrambled control (0.095 ± 0.001) and blank control groups (0.099 ± 0.001) (t = 10.227, 10.934, P < 0.01), which further supported the highest effectiveness of EgRad9-siRNA-614. The EgRad9 mRNA expression levels in the EgRad9-siRNA-354, -614, and -971 interference groups were 0.432 ± 0.055, 0.291 ± 0.079, and 0.612 ± 0.032, respectively, with significant differences between EgRad9-siRNA-614 versus scrambled control (1.001 ± 0.020) and blank control groups (1.001 ± 0.012) (t = 7.874, 7.663, P < 0.01), again verifying the highest effectiveness of EgRad9-siRNA-614. Under the same electrophoresis condition, the DNA fragments in the EgRad9-siRNA-614 group migrated from the nucleus to the anode, with a smearing tail. The Olive tail moment in the EgRad9-siRNA-614 group was 13.901 ± 2.263, significantly different from the scrambled control (0.074 ± 0.020) and blank control groups (0.047 ± 0.034) (t = 12.845, 13.251, P < 0.01). The ROS content in the EgRad9-siRNA-614 group was 13.024 ± 0.154, significantly different from the scrambled control (2.728 ± 0.083) and blank control groups (2.555 ± 0.007) (t = 9.296, 10.134, P < 0.01). Conclusion The EgRad9 gene plays an important role in DNA oxidative damage. The EgRad9-siRNA-614 can specifically interfere with the expression of EgRad9 in protoscoleces of Echinococcus granulosus, and reduce the ability of DNA oxidative damage repair in protoscoleces.

Key words: EgRad9 gene, siRNA, Echinococcus granulosus, DNA oxidative damage