siRNA特异性干扰EgTPx基因表达对细粒棘球蚴原头节DNA氧化损伤机制的影响
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摘要
目的研究小干扰RNA(small interfering RNA,siRNA)特异性干扰EgTPx基因表达后对细粒棘球蚴(Echi-nococcus granulosus,Eg)原头节DNA氧化损伤机制的影响。方法利用电穿孔法将EgTPx-siRNA干扰序列转染细粒棘球蚴原头节,实验分为EgTPx-siRNA-60、115、250干扰组,阴性对照组和空白对照组。电转3d后,协同自然状态下原头节体外耐受H_2O_2最大浓度干预1h,综合伊红拒染试验和碱性磷酸酶检测干扰前后原头节活性变化及qRT-PCR检测干扰前后EgTPx-mRNA表达水平变化筛选最佳EgTPx-siRNA干扰序列。采用单细胞凝胶电泳法(彗星试验)检测干扰前后原头节DNA氧化损伤程度变化;采用试剂盒法检测原头节体内活性氧(reactive oxygen species,ROS)含量,分析siRNA干扰对DNA氧化损伤机制的影响。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0.4mmol/L,干预时间为1h;电穿孔法将绿色荧光干扰序列导入原头节体内,实验组和阴性对照组的原头节体内均有绿色荧光斑点,而空白对照组原头节体内无绿色荧光斑点。导入siRNA后通过伊红拒染试验、碱性磷酸酶活性检测及qRT-PCR筛选出最佳干扰序列为EgTPx-siRNA-60;彗星试验显示干扰EgTPx基因表达后EgTPx-siRNA-60干扰组原头节DNA碎片离开核向阳极迁移形成拖尾,EgTPx-siRNA-60干扰组彗星尾距OTM值为12.860 2±2.537 7,与阴性对照组(0.055 8±0.010 3)和空白对照组(0.037 2±0.009 8)相比差异有统计学意义(t值分别为11.251和12.845,均P<0.01);经EgTPx-siRNA-60序列干扰后原头节体内ROS含量(13.978 6±0.233 0)A.U.与阴性对照组(3.281 7±0.052 5)A.U.和空白对照组(3.018 2±0.076 9)A.U.比较差异有统计学意义(t值分别为8.926和9.314,均P<0.01)。结论 EgTPx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,EgTPx-siRNA-60可特异性干扰EgTPx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 EgTPx gene expression on DNA oxidative damage in protoscoleces of Echinococcus granulosus. Methods EgTPx-siRNA was transfected into protoscoleces of E.granulosus by electroporation.Protoscoleces were divided into EgTPx-siRNA-60,EgTPx-siRNA-115,and EgTPx-siRNA-250 interference groups,a scrambled control group,and a blank control group.Three days after electroporation,the protoscoleces were treated with the maximum tolerated concentration of H_2O_2 for 1 h.Activity before and after transfection were compared using an eosin exclusion test,alkaline phosphatase was detected in protoscoleces,and the level of EgTPx-mRNA transcription was determined before and after transfection using qRT-PCR to screen for the most effective siRNA.Single cell gel electrophoresis was used to detect the extent of DNA damage before and after transfection.The level of reactive oxygen species(ROS)in protoscoleces was assessed using an ROS detection kit.Data were analyzed using the software SPSS 22.0. Results The maximum tolerated concentration of H_2O_2 for protoscoleces of E.granulosus was 0.4 mmol/L,and the duration of intervention was 1 h.The siRNA sequences were effectively transfected via 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 rate of protoscoleces was 32.82±3.29%in the EgTPx-siRNA-60 interference group,70.03±6.65%in the EgTPx-siRNA-115 interference group,and 55.46±6.01%in the EgTPx-siRNA-250 interference group.Thus,EgTPx-siRNA-60 was deemed to be the most effective interfering sequence.Alkaline phosphatase activity was 0.0 273±0.0 012 in the EgTPx-siRNA-60 interference group,0.0 645±0.0 058 in the EgTPxsiRNA-115 interference group,and 0.0 424±0.0 037 in the EgTPx-siRNA-250 interference group.The level of alkaline phosphatase activity in the EgTPx-siRNA-60 interference group differed significantly from the level in the scrambled control group(0.0 978±0.0 063)and blank control group(0.0 989±0.0 042)(t=9.257,9.946,P<0.01),which further substantiated the effectiveness of EgTPx-siRNA-60.The level of EgTPx-mRNA expression was 0.3 883±0.0 450 A.U.in the EgTPx-siRNA-60 interference group,0.7 416±0.0 309 A.U.in the EgTPx-siRNA-115 interference group,and 0.5 681±0.0 721 A.U.in the EgTPx-siRNA-250 interference group.The level of EgTPx-mRNA expression in the EgTPx-siRNA-60 interference group differed significantly from the level in the scrambled control group(1.0 099±0.0 105 A.U.)and blank control group(1.0 007±0.0 087 A.U.)(t=7.824,7.553,P<0.01),again verifying the effectiveness of EgTPx-siRNA-60.Under the same electrophoresis conditions,DNA fragments in the EgTPx-siRNA-60 group migrated from the nucleus to the anode,with a smeared tail.The Olive tail moment in the EgTPx-siRNA-60 group was 12.8 602±2.5 377,which differed significantly from that in the scrambled control group(0.0 558±0.0 103)and blank control group(0.0 372±0.0 098)(t=11.251,12.845,P<0.01).The level of ROS in the EgTPx-siRNA-60 group was 13.9 786±0.2 330 A.U.,which differed significantly from that in the scrambled control group(3.2 817±0.0 525 A.U.)and blank control group(3.0 182±0.0 769 A.U.)(t=8.926,9.314,P<0.01). Conclusion The EgTPx gene plays an important role in DNA oxidative damage.EgTPx-siRNA-60 specifically interferes with the expression of EgTPx in protoscoleces of E.granulosus,and it reduce the capacity for repair of DNA oxidative damage in protoscoleces.
Objective To investigate the effect of siRNA interference with EgTPx gene expression on DNA oxidative damage in protoscoleces of Echinococcus granulosus. Methods EgTPx-siRNA was transfected into protoscoleces of E.granulosus by electroporation.Protoscoleces were divided into EgTPx-siRNA-60,EgTPx-siRNA-115,and EgTPx-siRNA-250 interference groups,a scrambled control group,and a blank control group.Three days after electroporation,the protoscoleces were treated with the maximum tolerated concentration of H_2O_2 for 1 h.Activity before and after transfection were compared using an eosin exclusion test,alkaline phosphatase was detected in protoscoleces,and the level of EgTPx-mRNA transcription was determined before and after transfection using qRT-PCR to screen for the most effective siRNA.Single cell gel electrophoresis was used to detect the extent of DNA damage before and after transfection.The level of reactive oxygen species(ROS)in protoscoleces was assessed using an ROS detection kit.Data were analyzed using the software SPSS 22.0. Results The maximum tolerated concentration of H_2O_2 for protoscoleces of E.granulosus was 0.4 mmol/L,and the duration of intervention was 1 h.The siRNA sequences were effectively transfected via 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 rate of protoscoleces was 32.82±3.29%in the EgTPx-siRNA-60 interference group,70.03±6.65%in the EgTPx-siRNA-115 interference group,and 55.46±6.01%in the EgTPx-siRNA-250 interference group.Thus,EgTPx-siRNA-60 was deemed to be the most effective interfering sequence.Alkaline phosphatase activity was 0.0 273±0.0 012 in the EgTPx-siRNA-60 interference group,0.0 645±0.0 058 in the EgTPxsiRNA-115 interference group,and 0.0 424±0.0 037 in the EgTPx-siRNA-250 interference group.The level of alkaline phosphatase activity in the EgTPx-siRNA-60 interference group differed significantly from the level in the scrambled control group(0.0 978±0.0 063)and blank control group(0.0 989±0.0 042)(t=9.257,9.946,P<0.01),which further substantiated the effectiveness of EgTPx-siRNA-60.The level of EgTPx-mRNA expression was 0.3 883±0.0 450 A.U.in the EgTPx-siRNA-60 interference group,0.7 416±0.0 309 A.U.in the EgTPx-siRNA-115 interference group,and 0.5 681±0.0 721 A.U.in the EgTPx-siRNA-250 interference group.The level of EgTPx-mRNA expression in the EgTPx-siRNA-60 interference group differed significantly from the level in the scrambled control group(1.0 099±0.0 105 A.U.)and blank control group(1.0 007±0.0 087 A.U.)(t=7.824,7.553,P<0.01),again verifying the effectiveness of EgTPx-siRNA-60.Under the same electrophoresis conditions,DNA fragments in the EgTPx-siRNA-60 group migrated from the nucleus to the anode,with a smeared tail.The Olive tail moment in the EgTPx-siRNA-60 group was 12.8 602±2.5 377,which differed significantly from that in the scrambled control group(0.0 558±0.0 103)and blank control group(0.0 372±0.0 098)(t=11.251,12.845,P<0.01).The level of ROS in the EgTPx-siRNA-60 group was 13.9 786±0.2 330 A.U.,which differed significantly from that in the scrambled control group(3.2 817±0.0 525 A.U.)and blank control group(3.0 182±0.0 769 A.U.)(t=8.926,9.314,P<0.01). Conclusion The EgTPx gene plays an important role in DNA oxidative damage.EgTPx-siRNA-60 specifically interferes with the expression of EgTPx in protoscoleces of E.granulosus,and it reduce the capacity for repair of DNA oxidative damage in protoscoleces.
引文
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[11] Yang R,Wei L,Fu QQ,et al.SOD3 Ameliorates H2O2-in-duced oxidative damage in SH-SY5YCells by inhibiting the mito-chondrial pathway[J].Neurchem Res,2016,41(7):1818-30.
[12] Savina NV,Nikitchenko NV,Kuzhir TD,et al.The cellular re-sponse to oxidatively induced DNA damage and polymorphism ofsome DNA eepair genes associated with clinicopathological fea-tures of bladder cancer[J].Oxid Med Cell longe,2016,2016(7):5710403.
[13] Cucher M,Prada L,Mourglia-Ettlin G,et al.Identification ofEchinococcus granulosus microRNAs and their expression in dif-ferent life cycle stages and parasite genotypes[J].Int J Parasitol,2011,41(3-4):439-48.
[14] Yang M,Li J,Wu J,et al.Cloning and characterization of anEchinococcus granulosus ecdysteroid hormone nuclear receptorHR3-like gene[J].Parasite,2017(24):36.
[15] 高上吉,钟晓霞,刘婉莹,等.基于彗星实验的洛克沙胂对秀丽隐杆线虫胚胎细胞DNA损伤的研究[J].生态毒理学报,2016,11(3):167-72.
[16] 郑海亚,文丽梅,吕国栋,等.青蒿琥酯体外抗细粒棘球蚴活性氧对DNA作用机制影响的研究[J].中国病原生物学杂志,2017,12(8):751-6.
[2] Redza-Dutordoir M,Averill-Bates DA.Activation of a apotosissignaling pathways by reactive oxygen species[J].Biochim Bio-phys Acta,2016,1863(12):2977-92.
[3] Diwanji N,Bergmann A.The beneficial role of extracellular reac-tive oxygen species in apoptosis-induced compensatory prolifera-tion[J].Fly(Austin),2017,11(1):46-52.
[4] Zhang NZ,Liu JY,Li WH,et al.Cloning and characterization ofthioredoxin peroxidases fromTrichinella spiralis[J].Vet Parasi-tol,2016(231):53.
[5] 王慧,李军,张富春,等.细粒棘球绦虫硫氧化蛋白过氧化物酶基因在毕赤酵母中的分泌表达及生物学功能鉴定[J].中国病原生物学杂志,2014,9(3):220-4.
[6] Salinas G,Frenandez V,Frenandez C,et al.Echinococcus gran-ulosus:cloning of a thioredoxin peroxidase[J].Exp Parasitol,1998,90(3):298-301.
[7] Li J,Zhang WB,Loukas A,et al.Functional expression andcharacterization of Echinococcus granulosus thioredoxin peroxi-dase suggests a role in protection against oxidaticve damage[J].Gene,2004(326):157-65.
[8] 肖云峰.细粒棘球绦虫原头蚴在青蒿素干预下的基因转录谱分析[D].乌鲁木齐:新疆医科大学,2014.
[9] Pierson L,Mousley A,Devine L,et al.RNA interference in acestode reveals specific silencing of selected highly expressed genetranscripts[J].Int J Parasitol,2010,40(5):605-15.
[10] Mizukami C,Spiliotis M,Gottstein B,et al.Gene silencing inEchinococcus multilocularis protoscoleces using RNA interference[J].Parasitol Int,2010,59(4):647-52.
[11] Yang R,Wei L,Fu QQ,et al.SOD3 Ameliorates H2O2-in-duced oxidative damage in SH-SY5YCells by inhibiting the mito-chondrial pathway[J].Neurchem Res,2016,41(7):1818-30.
[12] Savina NV,Nikitchenko NV,Kuzhir TD,et al.The cellular re-sponse to oxidatively induced DNA damage and polymorphism ofsome DNA eepair genes associated with clinicopathological fea-tures of bladder cancer[J].Oxid Med Cell longe,2016,2016(7):5710403.
[13] Cucher M,Prada L,Mourglia-Ettlin G,et al.Identification ofEchinococcus granulosus microRNAs and their expression in dif-ferent life cycle stages and parasite genotypes[J].Int J Parasitol,2011,41(3-4):439-48.
[14] Yang M,Li J,Wu J,et al.Cloning and characterization of anEchinococcus granulosus ecdysteroid hormone nuclear receptorHR3-like gene[J].Parasite,2017(24):36.
[15] 高上吉,钟晓霞,刘婉莹,等.基于彗星实验的洛克沙胂对秀丽隐杆线虫胚胎细胞DNA损伤的研究[J].生态毒理学报,2016,11(3):167-72.
[16] 郑海亚,文丽梅,吕国栋,等.青蒿琥酯体外抗细粒棘球蚴活性氧对DNA作用机制影响的研究[J].中国病原生物学杂志,2017,12(8):751-6.
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