Zinc Finger Nucleases Targeting the Human Papillomavirus E7 Oncogene Induce E7 Disruption and a Transformed Phenotype in HPV16/18-Positive Cervical Cancer Cells
Zinc Finger Nucleases Targeting the Human Papillomavirus E7 Oncogene Induce E7 Disruption and a Transformed Phenotype in HPV16/18-Positive Cervical Cancer Cells
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靶向人乳头瘤病毒 E7 癌基因的锌指核酸酶在 HPV16/18 阳性宫颈癌细胞中诱导 E7 破坏和表型转变
DOI：
10.1158/1078-0432.ccr-14-0250
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发表时间：
2014-12-15
影响因子：
11.5
通讯作者：
Wang, Hui
中科院分区：
文献类型：
--
作者：
Ding, Wencheng;Hu, Zheng;Wang, Hui
目的：宫颈癌主要由高危型人乳头瘤病毒（HR - HPV）感染引起。HR - HPV致癌基因E6和E7的持续表达与恶性转化有关。本研究旨在提供概念验证数据，以支持使用靶向HPV E7的锌指核酸酶（ZFN）治疗HPV相关宫颈癌。
实验设计：我们设计并构建了能够特异性识别和切割HPV16/18 E7 DNA的ZFN。我们通过单链退火（SSA）试验检测了所选的ZFN16 - E7 - S2和ZFN18 - E7 - S2的切割效率。通过细胞活力和集落形成试验评估接受ZFN治疗的细胞生长抑制情况。通过蛋白质印迹法检测HPV E7及下游基因的基因破坏情况。通过细胞凋亡试验检测诱导HPV型特异性凋亡的特异性和效率。我们还引入了异种移植形成试验以评估抑制HPV相关疾病的潜力。
结果：我们发现ZFN16 - E7 - S2和ZFN18 - E7 - S2破坏了HPV16/18阳性宫颈癌细胞中的HPV E7致癌基因。这两种ZFN均有效抑制了特定类型宫颈癌细胞的生长，并特异性诱导了相应的HPV16和HPV18阳性宫颈癌细胞系的凋亡。ZFN16 - E7 - S2和ZFN18 - E7 - S2在体内也抑制了异种移植瘤的形成。
结论：靶向HPV16/18 E7的ZFN可有效诱导E7致癌基因的破坏，并导致HPV阳性细胞的类型特异性和高效生长抑制及凋亡。靶向HPV16/18 E7致癌基因的ZFN可作为治疗HPV相关宫颈癌的新型治疗剂。（C）2014美国癌症研究协会
Purpose: Cervical cancer is mainly caused by infections of high-risk human papillomavirus (HR-HPV). Persistent expression of HR-HPV oncogenes E6 and E7 is implicated in malignant transformation. The aim was to provide proof-of-concept data to support use of zinc finger nucleases (ZFN) targeting HPV E7 to treat HPV-related cervical cancer.Experimental Design: We designed and constructed ZFNs that could specifically recognize and cleave HPV16/18 E7 DNA. We tested the cleavage efficiency of selected ZFN16-E7-S2 and ZFN18-E7-S2 by using single-strand annealing (SSA) assay. Cell viability and colony formation assays were used to estimate the inhibition of cell growth that received treatments of ZFNs. Gene disruption of HPV E7 and downstream genes were examined by Western blotting. Cell apoptosis assay was used to test the specificity and efficiency of induction of HPV type-specific apoptosis. We also introduced xenograft formation assays to estimate the potential of inhibition of HPV-related disease.Results: We found ZFN16-E7-S2 and ZFN18-E7-S2 disrupted HPV E7 oncogenes in HPV16/18-positive cervical cancer cells. Both ZFNs effectively led to inhibition of type-specific cervical cancer cell growth, and specifically induced apoptosis of corresponding HPV16-and HPV18-positive cervical cancer cell lines. ZFN16-E7-S2 and ZFN18-E7-S2 also repressed xenograft formation in vivo.Conclusion: ZFNs targeting HPV16/18 E7 could effectively induce disruption of E7 oncogenes and lead to type-specific and efficient growth inhibition and apoptosis of HPV-positive cells. ZFNs targeting HPV16/ 18 E7 oncogenes could be used as novel therapeutic agents for the treatment of HPV-related cervical cancer. (C) 2014 AACR.