Effect and mechanism of CRISPR/Cas system on proliferation and apoptosis of human cervical cancer cells

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

Abstract

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

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.

References

［1］ Wiedenheft B, Sternberg SH, Doudna JA. RNAguided genetic silencing systems in bacteria and archaea［J］. Nature, 2012, 482(7385): 331-338. ［2］ Makarova KS, Aravind L, Wolf YI, et al. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPRCas systems［J］. Biol Direct, 2011, 6: 38. ［3］ Cho SW, Kim S, Kim JM, et al. Targeted genome engineering in human cells with the Cas9 RNAguided endonuclease［J］. Nat Biotechnol, 2013, 31(3): 230-232. ［4］ Mali P, Yang LH, Esvelt KM, et al. RNAGuided human genome engineering via Cas9［J］. Science, 2013, 339(6121): 823-826. ［5］ Mahfouz MM, Li L, Shamimuzzaman M, et al. De novoengineered transcription activatorlike effector (TALE) hybrid nuclease with novel DNA binding specificity creates doublestrand breaks［J］. Proc Natl Acad Sci USA, 2011, 108(6): 2623-2628. ［6］ Chung CH, Gillison ML. Human papillomavirus in head and neck cancer: its role in pathogenesis and clinical implications［J］. Clin Cancer Res, 2009, 15(22): 6758-6762. ［7］ Jonson AL, Rogers LM, Ramakrishnan S, et al. Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer［J］. Gynecol Oncol, 2008, 111(2): 356-364. ［8］彭婵娟，叶枫，谢幸． RNA干扰在宫颈癌HPV16 E6/E7中的研究进展［J］．国际肿瘤学杂志， 2011， 38（4）： 300-303．［9］ Katic I, Groβhans H. Targeted heritable mutation and gene conversion by Cas9CRISPR in Caenorhabditis elegans［J］. Genetics, 2013, 195(3): 1173-1176. ［10］ Hwang WY, Fu Y, Reyon D, et al. Efficient genome editing in zebrafish using a CRISPRCas system［J］. Nat Biotechnol, 2013, 31(3): 227-229. ［11］ Xue W, Chen S, Yin H, et al. CRISPRmediated direct mutation of cancer genes in the mouse liver［J］. Nature, 2014, 514(7522): 380-384. ［12］ Yoshimi K, Kaneko T, Voigt B, et al. Allelespecific genome editing and correction of diseaseassociated phenotypes in rats using the CRISPRCas platform［J］. Nat Commun, 2014, 5: 4240. ［13］ Wang HY, Yang H, Shivalila CS, et al. Onestep generation of mice carrying mutations in multiple genes by CRISPR/Casmediated genome engineering［J］. Cell, 2013, 153(4): 910-918. ［14］ Fu Y, Foden JA, Khayter C, et al. Highfrequency offtarget mutagenesis induced by CRISPRCas nucleases in human cells［J］. Nat Biotechnol, 2013, 31(9): 822-826. ［15］ Ran FA, Hsu PD, Lin CY, et al. Double nicking by RNAguided CRISPR Cas9 for enhanced genome editing specificity［J］. Cell, 2013, 154(6): 1380-1389.

[1]	Zhang Ningning, Yang Zhe, Tan Limei, Li Zhenning, Wang Di, Wei Yongzhi. Diagnostic value of cervical cell DNA ploidy analysis combined with B7-H4 and PKCδ for cervical cancer [J]. Journal of International Oncology, 2024, 51(5): 286-291.
[2]	Zhang Lu, Jiang Hua, Lin Zhou, Ma Chenying, Xu Xiaoting, Wang Lili, Zhou Juying. Analysis of curative effect and prognosis of immune checkpoint inhibitor in the treatment of recurrent and metastatic cervical cancer [J]. Journal of International Oncology, 2023, 50(8): 475-483.
[3]	Lyu Lu, Sun Pengfei. Gut flora and cervical cancer [J]. Journal of International Oncology, 2023, 50(6): 373-376.
[4]	Ma Xueyan, Lu Lili, Sun Pengfei. Advances in the immune microenvironment in cervical cancer [J]. Journal of International Oncology, 2023, 50(1): 47-50.
[5]	Zhang Lu, Zhou Juying, Ma Chenying, Lin Zhou. Advances in immunotherapy for recurrent and metastatic cervical cancer [J]. Journal of International Oncology, 2022, 49(9): 517-520.
[6]	Shi Yingxia, Hu Lijun, Yu Jingping. Application of immune checkpoint inhibitors in the treatment of recurrent or metastatic cervical cancer [J]. Journal of International Oncology, 2022, 49(9): 568-571.
[7]	Peng Chen, Xie Yintong, Zhang Xin, Xie Peng. Research progress of maintenance therapy for cervical cancer [J]. Journal of International Oncology, 2022, 49(7): 430-435.
[8]	Yuan Chenyang, Zhou Juying. Research progress on prognostic factors of cervical cancer [J]. Journal of International Oncology, 2022, 49(5): 307-313.
[9]	Wang Yue, Wu Qiong, Xu Yuan, Gong Wei, Xu Xiaoting. Screening and treatment progression of elderly cervical cancer [J]. Journal of International Oncology, 2022, 49(12): 754-758.
[10]	Ma Xiuzhen, Lu Yan, Zhao Bingbing, Qiu Hongcong, Xu Xun, Wei Min. Effects of total flavonoids from Baeckea frutescens on the migration, invasion and apoptosis of cervical cancer SiHa cells [J]. Journal of International Oncology, 2021, 48(4): 206-211.
[11]	Yu Mingyue, Chen Zhengzheng, Zhao Xuxu, Ren Pingping, Zhang Ying, Ge Li, Zhu Meiling, Zhao Weidong. Factors related to postoperative adjuvant therapy of locally advanced cervical cancer and building of a nomogram prediction model [J]. Journal of International Oncology, 2021, 48(1): 35-40.
[12]	Ding Xuchao, Cao Lili. Common active targeting nano drug delivery systems for cervical cancer [J]. Journal of International Oncology, 2021, 48(1): 61-64.
[13]	Shu Hang, Xu Zhonghua, Zhu Haochen, Yang Yahui, Lyu Yin. Research progress of radiosensitivity in cervical cancer [J]. Journal of International Oncology, 2020, 47(8): 496-500.
[14]	Zheng Xin, Chen Xiaopin. Relationship between HPV and oropharyngeal cancer in China [J]. Journal of International Oncology, 2020, 47(3): 164-168.
[15]	Song Mingze, Cheng Yiming, Li Gang, Wang Zhenming, Li Shirong. Value of P16/Ki-67 double staining detection in screening cervical cancer and precancerous lesions [J]. Journal of International Oncology, 2020, 47(11): 675-681.