CRISPR/Cas系统对人宫颈癌细胞增殖与凋亡的影响及其机制探讨

doi:10.3760/cma.j.issn.1673-422X.2015.12.002

摘要/Abstract

摘要： 目的研究靶向于HPV16型的E7（HPV16E7）基因的成簇规律间隔短回文重复序列及其相关系统（CRISPR/Cas）对HPV16阳性宫颈癌细胞株SiHa增殖和凋亡的影响及其机制。方法培养HPV16阳性的宫颈癌细胞株SiHa和HPV阴性的人胚肾上皮细胞株HEK293，分别将SiHa细胞和HEK293细胞分为3组：Cri组（按照1∶3的比例转染向导RNA与Cas9质粒）、C9组（只转染等量的Cas9质粒）和对照组（不处理）。T7核酸内切酶Ⅰ法检测双链DNA断裂情况，流式细胞术检测细胞凋亡，CCK8法评价细胞增殖，Western blotting检测E7和成视网膜母细胞瘤蛋白（pRb）表达。结果转染48 h后，SiHa细胞Cri组PCR产物被切开。SiHa细胞Cri组凋亡率为26.6%，明显高于对照组（2.6%，χ2=5.455，P=0.020）和C9组（3.1%，χ2=6.279，P=0.012）。HEK293细胞对照组、C9组和Cri组的凋亡率分别为7.4%、7.6%、7.9%，Cri组与对照组和C9组比较，差异均无统计学意义（χ2=0.032，P=0.858；χ2=0.034，P=0.853）。转染72 h后，SiHa细胞Cri组增殖抑制率为29.4%，明显高于对照组（15.0%，χ2=22.481，P=0.000）和C9组（18.0%，χ2=24.879，P=0.000）。Cri组HEK293细胞在72 h的增殖抑制率为3.2%，与C9组（2.2%，χ2=2.857，P=0.091）和对照组（2.3%，χ2=3.438，P=0.064）比较差异均无统计学意义。转染48 h后，与对照组相比，SiHa细胞Cri组E7蛋白表达明显下调，pRb蛋白表达明显上升，而C9组E7蛋白和pRb蛋白含量没有明显变化。结论靶向HPV16E7的CRISPR可以特异性地促进HPV16阳性的SiHa细胞凋亡、抑制细胞增殖。其机制可能是通过特异性地作用于HPV16E7基因使其双链DNA断裂、功能破坏，同时上调抑癌蛋白pRb表达。

关键词: 宫颈肿瘤, 人乳头瘤病毒16, 成簇规律间隔短回文重复序列及其相关系统

Abstract: ObjectiveTo investigate the effect and mechanism of HPV16E7 gene specific CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR associated system) on the cell apoptosis and proliferation of human cervical cancer SiHa cell line. MethodsHPV16 positive cervical cancer SiHa cells and HPV16 negative human embryonic kidney HEK293 cells were cultured and each of the cells were divided into 3 groups respectively in the experiment: the control group was untreated, the C9 group was transfected only with Cas9 plasmid, and the Cri group was cotransfected with guide RNA (gRNA) plasmid and Cas9 plasmid (1∶3). T7 endonuclease Ⅰ assay was used to detect double strand break (DSB) formation in SiHa cells. The apoptosis rates of SiHa and HEK293 cells were detected by flow cytometry (FCM). CCK8 was used to evaluate the proliferation of SiHa and HEK293 cells. Western blotting was applied to detect E7 and pRb protein expression. ResultsThe SiHa cells in Cri group showed DSB formation at 48 h after transfection. The apoptosis rate of Cri group at 48 h was 26.6%, higher than 2.6% in control group (χ2=5.455, P=0.020) and 3.1% in C9 group (χ2=6.279, P=0.012). The apoptosis rates of control, C9 and Cri group were 7.4%, 7.6%, 7.9% for HEK293 cells respectively. Compared with the control and C9 group, the apoptosis rate of the Cri group showed no statistical significance （χ2=0.032, P=0.858; χ2=0.034, P=0.853）. After 72 h transfection, the inhibition rate of SiHa cells in Cri group was 29.4%, higher than 15.0% in control group (χ2=22.481, P=0.000) and 18.0% in C9 group (χ2=24.879, P=0.000). The inhibition rate of the HEK293 cells in Cri group was 3.2%, and it showed no significant difference compared with the inhibition rate of C9 group (2.2%, χ2=2.857, P=0.091) and control group (2.3%, χ2=3.438, P=0.064) respectively. Downregulated expression of E7 protein and upregulated expression of pRb protein were detected of the SiHa cells in Cri group compared with the control group, while the E7 protein and pRb protein level did not show difference in the C9 group. ConclusionCRISPR specifically targeting HPV16E7 oncogene can promote apoptosis of SiHa cells, and inhibit the cell proliferation. It is speculated that CRISPR system may induce DSB event of E7 gene, and result into increased expression of tumor suppressor protein pRb.

Key words: Uterine cervical neoplasms, Human papillomavirus 16, Clustered regularly interspaced short palindromic repeats/CRISPR associated system

王瑞玲,胡争,夏薇,魏波,叶望莲,朱红芳. CRISPR/Cas系统对人宫颈癌细胞增殖与凋亡的影响及其机制探讨[J]. 国际肿瘤学杂志, 2015, 42(12): 886-890.

Wang Ruiling, Hu Zheng, Xia Wei, Wei Bo, Ye Wanglian, Zhu Hongfang. Effect and mechanism of CRISPR/Cas system on proliferation and apoptosis of human cervical cancer cells[J]. Journal of International Oncology, 2015, 42(12): 886-890.

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