siRNA特异性干扰EgRad9基因表达对细粒棘球蚴原头节DNA氧化损伤机制的影响

Effect of siRNA interference with EgRad9 gene on DNA oxidative damage in protoscoleces of Echinococcus granulosus

目的研究小干扰RNA(small interfering RNA,si RNA)特异性干扰Eg Rad9基因表达后对细粒棘球蚴原头节DNA氧化损伤机制的影响。方法利用电穿孔法将Eg Rad9-si RNA干扰序列和阴性对照干扰序列转染细粒棘球蚴原头节,实验分为Eg Rad9-si RNA-354、-614、-971干扰组,阴性对照组和空白对照组。电转染3 d后,加入自然状态下原头节最大耐受浓度的H_2O_2处理1 h,伊红染液染色后,倒置荧光显微镜下观察原头节活性并计算存活率。收集各组原头节的培养液,进行碱性磷酸酶活性的检测。电转染3 d后,加入自然状态下原头节耐受H_2O_2的最大耐受浓度处理1 h后,TRIzol法提取总RNA。实时荧光定量PCR(q RT-PCR)检测Eg Rad9m RNA的表达。采用彗星实验检测干扰Eg Rad9基因表达后细粒棘球蚴原头节DNA氧化损伤情况,活性氧(reactive oxygen species,ROS)检测试剂盒检测干扰Eg Rad9基因表达后细粒棘球蚴原头节体内ROS的含量,采用SPSS 22.0统计学软件进行数据分析。结果自然状态下细粒棘球蚴原头节体外耐受H_2O_2的最大浓度为0.4 mmol/L,干预时间为1 h;电穿孔法可将绿色荧光干扰序列有效的导入原头节体内,实验组和阴性对照组的原头节体内均有明亮的绿色荧光斑点;而空白对照组原头节体内无绿色荧光斑点。Eg Rad9-si RNA-354、-614、-971干扰组原头节的存活率分别为(42.99±4.03)%、(29.86±5.87)%和(56.48±4.64)%,初步筛选最有效干扰序列为Eg Rad9-si RNA-614。si RNA-354、-614、-971干扰组碱性磷酸酶活性分别为0.024±0.001、0.004±0.001、0.039±0.002,其中Eg Rad9-si RNA-614组碱性磷酸酶活性与阴性对照组(0.095±0.001)和空白对照组(0.099±0.001)相比,差异均有统计学意义(t=10.227、10.934,均P<0.01),表明该组原头节Eg Rad9基因经干扰后对其活性影响最大,最有效干扰序列为Eg Rad9-si RNA-614。Eg Rad9-si RNA-354、614、971干扰组Eg Rad9 m RNA表达量分别为0.432±0.055、0.291±0.079、0.612±0.032,其中经Eg Rad9-si RNA-614序列干扰后,Eg Rad9 m RNA表达量与阴性对照组(1.001±0.020)和空白对照组(1.001±0.012)相比均显著下调(t=7.874、7.663,均P<0.01),可确定最有效干扰序列为Eg Rad9-si RNA-614。相同电泳条件下,Eg Rad9-si RNA-614干扰组原头节DNA碎片离开核向阳极迁移,形成拖尾;Eg Rad9-si RNA-614干扰组彗星尾距OTM值为13.901±2.263,与阴性对照组(0.074±0.020)和空白对照组(0.047±0.034)相比,差异有统计学意义(t=12.845、13.251,均P<0.01)。经Eg Rad9-si RNA-614序列干扰后,原头节体内ROS含量为13.024±0.154,与阴性对照组(2.728±0.083)和空白对照组(2.555±0.007)相比,差异有统计学意义(t=9.296、10.134,均P<0.01)。结论Eg Rad9基因在细粒棘球蚴原头节DNA氧化损伤过程中发挥重要作用,Eg Rad9-si RNA-614干扰片段可特异性干扰Eg Rad9基因的表达,并降低原头节修复DNA氧化损伤的能力。

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 i RNA 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 protoscole-ces 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 q RT-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 clear-ly 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.