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

中国寄生虫学与寄生虫病杂志 ›› 2018, Vol. 36 ›› Issue (4): 343-349.

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

郑璇^1,², 卢帅², 赵军², 吕国栋³, 文丽梅^2,⁴, 李亚芬^1,², 田春艳^1,², 王建华^2,^*()

新疆医科大学,1 药学院
2 第一附属医院药学部临床药学科
3 第一附属医院临床医学研究院,新疆重大疾病医学重点实验室-省部共建国家重点实验室培育基地
4 第一附属医院,省部共建中亚高发病成因与防治国家重点实验室,乌鲁木齐 830054

收稿日期:2018-01-08 出版日期:2018-08-30 发布日期:2018-09-06
通讯作者: 王建华
基金资助:
国家自然科学基金（No. 81560607）;省部共建中亚高发病成因与防治国家重点实验室项目（No. SKL-HIDCA-2017-Y7）;新疆维吾尔自治区药学会项目（No. YXH201704）

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

Xuan ZHENG^1,², Shuai LU², Jun ZHAO², Guo-dong LV³, Li-mei WEN^2,⁴, Ya-fen LI^1,², Chun-yan TIAN^1,², Jian-hua WANG^2,^*()

Xinjiang Medical University, 1 College of Pharmaceutical Sciences
2 Clinical Pharmacy of the First Affiliated Hospital
3 State Key Laboratory Incubation Base of Xinjiang Major Diseases Research, Clinical Medical Research Institute, the First Affiliated Hospital
4 State Key Laboratory of Pathogenesis, Prevention, Treatment of Central Asian High Incidence Diseases, the First Affiliated Hospital, Urumqi 830054, China

Received:2018-01-08 Online:2018-08-30 Published:2018-09-06
Contact: Jian-hua WANG
Supported by:
Supported by the National Natural Science Foundation of China(No. 81560607), the Ministry-Provincial Cooperation for the Establishment of the State Key Laboratory on the Causes and Control of High Incidence of Disease in Central Asia(No. SKL-HIDCA-2017-Y7), and the Project of Xinjiang Uygur Autonomous Region Medical Association(No. YXH201704)

摘要/Abstract

摘要：

目的研究小干扰RNA（small interfering RNA,siRNA）特异性干扰EgRad9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EgRad9-siRNA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EgRad9-siRNA-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H₂O₂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H₂O₂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（qRT-PCR）检测EgRad9 mRNA的表达。采用彗星实验检测干扰EgRad9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EgRad9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H₂O₂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。EgRad9-siRNA-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EgRad9-siRNA-614。siRNA-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EgRad9-siRNA-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EgRad9基因经干扰后对其活性影响最大,最有效干扰序列为EgRad9-siRNA-614。EgRad9-siRNA-354、614、971干扰组EgRad9 mRNA表达量分别为0.432 ± 0.055、0.291 ± 0.079、0.612 ± 0.032,其中经EgRad9-siRNA-614序列干扰后,EgRad9 mRNA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EgRad9-siRNA-614。相同电泳条件下,EgRad9-siRNA-614干扰组原头节DNA碎片离开核向阳极迁移,形成拖尾;EgRad9-siRNA-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EgRad9-siRNA-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 EgRad9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,EgRad9-siRNA-614干扰片段可特异性干扰EgRad9基因的表达,并降低原头节修复DNA氧化损伤的能力。

关键词: EgRad9基因, 小干扰RNA, 细粒棘球蚴原头节, DNA氧化损伤

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

中图分类号:

R383.33

郑璇, 卢帅, 赵军, 吕国栋, 文丽梅, 李亚芬, 田春艳, 王建华. 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[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 343-349.

Xuan ZHENG, Shuai LU, Jun ZHAO, Guo-dong LV, Li-mei WEN, Ya-fen LI, Chun-yan TIAN, Jian-hua WANG. Effect of siRNA interference with EgRad9 gene on DNA oxidative damage in protoscoleces of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(4): 343-349.

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参考文献 17

[1]	Agudelo Higuita NI, Brunetti E, McCloskey C. Cystic echino-coccosis[J]. J Clin Microbiol, 2016, 54(3): 518-523.
[2]	Nakao M, Lavikainen A, Yanagida T, et al. Phylogenetic systema-tics of the genus Echinococcus(Cestoda ∶ Taeniidae)[J]. Int J Parasitol, 2013, 43(12-13): 1017-1029.
[3]	李永光, 付宝权. 寄生性蠕虫过氧化物氧还蛋白研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(1): 62-66.
[4]	Panieri E, Gogvadze V, Norberg E, et al. Reactive oxygen species generated in different compartments induce cell death, survival, or senescence[J]. Free Radic Biol Med, 2013, 57: 176-187.
[5]	Zhang Z, Cai Z, Li K, et al. The effect of ionizing radiation on mRNA levels of the DNA damage response genes rad9, rad1 and hus1 in various mouse tissues[J]. Radiat Res, 2015, 183(1): 94-104.
[6]	Tsai FL, Kai M.The checkpoint clamp protein Rad9 facilitates DNA-end resection and prevents alternative non-homologous end joining[J]. Cell Cycle, 2014, 13(21): 3460-3464.
[7]	Ghandhi SA, Ponnaiya B, Panigrahi SK, et al. RAD9 deficiency enhances radiation induced bystander DNA damage and transcriptomal response[J]. Radiat Oncol, 2014, 9: 206.
[8]	孙金帅, 陈志燕, 刘博, 等. Rad9在DNA损伤修复及细胞周期监测点调控中的作用[J]. 生命的化学, 2015, 6: 747-750.
[9]	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.
[10]	Pierson L, Mousley A, Devine L, et al. RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts[J]. Int J Parasitol, 2010, 40(5): 605-615.
[11]	Mizukami C, Spiliotis M, Gottstein B, et al. Gene silencing in Echinococcus multilocularis protoscoleces using RNA interference[J]. Parasitol Int, 2010, 59(4): 647-652.
[12]	康金凤, 胡汉华, 白山别克, 等. 过氧化氢体外诱导细粒棘球蚴原头节细胞凋亡的实验观察[J]. 中国寄生虫学与寄生虫病杂志, 2008, 26(5): 332-337.
[13]	Razavi-Azarkhiavi K, Behravan J, Mosaffa F, et al. Protective effects of aqueous and ethanol extracts of rosemary on H2O2-induced oxidative DNA damage in human lymphocytes by comet assay[J]. J Complement Integr Med, 2014, 11(1): 27-33.
[14]	Cucher M, Prada L, Mourglia-Ettlin G, et al. Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes[J]. Int J Parasitol, 2011, 41(3/4): 439-448.
[15]	Yang M, Li J, Wu J, et al. Cloning and characterization of an Echinococcus granulosus ecdysteroid hormone nuclear receptor HR3-like gene[J]. Parasite, 2017, 24: 36.
[16]	Collins AR.Measuring oxidative damage to DNA and its repair with the comet assay[J]. Biochim Biophys Acta, 2014, 1840(2): 794-800.
[17]	Lesgards JF, Gauthier C, Iovanna J, et al. Effect of reactive oxygen and carbonyl species on crucial cellular antioxidant enzymes[J]. Chem Biol Interact, 2011, 190(1): 28-34.

名称 Name	序列（5′→3′） Sequences (5′→3′)
EgRad9-siRNA-354	GCCUAGAAACUGGUCACAATT
EgRad9-siRNA-354	UUGUGACCAGUUUCUAGGCTT
EgRad9-siRNA-614	GCUGCUGUCAUUACGCAUATT
EgRad9-siRNA-614	UAUGCGUAAUGACAGCAGCTT
EgRad9-siRNA-971	CCGGCUUUAAAUACCACUUTT
EgRad9-siRNA-971	AAGUGGUAUUUAAAGCCGGTT
阴性对照Negative control	UUCUCCGAACGUGUCACGUTT
阴性对照Negative control	ACGUGACACGUUCGGAGAATT