HPV16E7靶向小干扰RNA对SiHa细胞增殖凋亡及6种抑癌基因的影响

中华皮肤科杂志 ›› 2016, Vol. 49 ›› Issue (10): 717-721.

HPV16E7靶向小干扰RNA对SiHa细胞增殖凋亡及6种抑癌基因的影响

龙嘉¹,²,李黎明³,许翠³,杨佳³,蒋明军⁴

1. 深圳大学附属第一医院深圳市第二人民医院
2. 中国医学科学院、北京协和医科大学皮肤病研究所
3. 中国医学科学院北京协和医学院皮肤病医院
4. 南京中国医学科学院北京协和医学院皮肤病研究所

收稿日期:2016-03-02 修回日期:2016-05-22 出版日期:2016-10-15 发布日期:2016-09-30
通讯作者: 蒋明军 E-mail:drmingjunjiang@163.com
基金资助:
国家自然科学基金;江苏省临床医学研究中心项目;中央级公益性科研院所基金科研业务费

Effects of a small interfering RNA targeting HPV16E7 on proliferation and apoptosis of SiHa cells and s of six tumor suppressor genes

Jia Long¹,², ³, ³,jia YANG³,Mingjun Jiang

Received:2016-03-02 Revised:2016-05-22 Online:2016-10-15 Published:2016-09-30
Contact: Mingjun Jiang E-mail:drmingjunjiang@163.com

摘要/Abstract

摘要：

目的探讨HPV16E7在SiHa细胞株中对抑癌基因（MT1G、NMES1、RRAD、SFRP1、SPARC、TFPI2）的表达及细胞增殖能力和凋亡水平的影响。方法 SiHa细胞转染E7SiRNA 48 h后，qPCR检测E7及6种抑癌基因 mRNA水平变化，CCK?8检测细胞增殖能力改变，流式细胞仪检测细胞凋亡水平。结果 SiHa细胞转染后48 h，qPCR结果显示实验组E7 mRNA显著降低（0.25 ± 0.036，P < 0.05）；6种抑癌基因的mRNA水平均显著增加（MT1G 1.403 ± 0.190、NMES1 1.720 ± 0.060、RRAD 1.390 ± 0.160、SFRP1 1.493 ± 0.120、SPARC 2.157 ± 0.144、TFPI2 2.060 ± 0.122，P < 0.05）。细胞增殖结果显示，与阴性对照组及空白组比较，实验组细胞增殖能力显著降低（0.554 ± 0.130，P <0.05），阴性对照组和空白组之间差异无统计学意义（P > 0.05）。细胞凋亡结果提示，实验组细胞凋亡细胞频率为9.222% 较阴性对照组（0.246%）及空白组（0.123%）明显增加（P < 0.05），空白组与阴性对照组差异无统计学意义（P > 0.05）。结论 HPV16导致细胞恶性转化过程中，E7可能通过抑制（MT1G、NMES1、RRAD、SFRP1、SPAR、TFPI2）6种抑癌基因的表达参与作用。

Abstract:

Long Jia, Li Liming, Xu Cui, Yang Jia, Jiang Mingjun Department of Dermatology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University，Shenzhen 518035, China （Long J）; Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College; Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Li LM, Xu C, Yang J, Jiang MJ) Corresponding author: Jiang Mingjun, Email: drmingjunjiang@163.com 【Abstract】 Objective To evaluate effects of human papilloma virus （HPV） 16E7 on s of six tumor suppressor genes （including MT1G, NMES1, RRAD, SFRP1, SPARC and TFPI2） in a cell line SiHa, as well as on its proliferation and apoptosis. Methods SiHa cells were divided into two groups to be transfected with a small interfering RNA targeting HPV16E7 （E7SiRNA, experimental group） and an empty vehicle （negative control group） respectively, with SiHa cells receiving no treatment serving as the blank control group. After 48 hours, qPCR was performed to measure the mRNA s of E7 and six tumor suppressor genes, CCK-8 assay to evaluate cellular proliferative activity, and flow cytometry to assess apoptosis of SiHa cells. Results At 48 hours after the transfection, the experimental group showed significantly decreased E7 mRNA （0.25 ± 0.036, P < 0.05）, but increased mRNA s of the six genes （MT1G 1.403 ± 0.190, NMES1 1.720 ± 0.060, RRAD 1.390 ± 0.160, SFRP1 1.493 ± 0.120, SPARC 2.157 ± 0.144, TFPI2 2.060 ± 0.122, all P < 0.05）. The proliferative activity of SiHa cells was significantly decreased （0.554 ± 0.130 vs. 1.028 ± 0.236 and 1.220 ± 0.126, both P < 0.05）, but the apoptosis rate was significantly increased （9.222% vs. 0.246% and 0.123%, both P < 0.05） in the experimental group compared with the negative control group and blank control group. No significant differences were observed between the negative control group and blank control group in proliferative activity or apoptosis rate of SiHa cells （both P > 0.05）. Conclusion E7 may participate in HPV16-induced cellular malignant transformation by suppressing the mRNA s of 6 tumor suppressor genes, including MT1G, NMES1, RRAD, SFRP1, SPAR and TFPI2.

龙嘉李黎明许翠杨佳蒋明军. HPV16E7靶向小干扰RNA对SiHa细胞增殖凋亡及6种抑癌基因的影响[J]. 中华皮肤科杂志, 2016, 49(10): 717-721.

Jia Long jia YANG Mingjun Jiang. Effects of a small interfering RNA targeting HPV16E7 on proliferation and apoptosis of SiHa cells and s of six tumor suppressor genes[J]. Chinese Journal of Dermatology, 2016, 49(10): 717-721.

参考文献 11

［1］	Au YCL, Tsang WP, Tsang TY, et al. HPV⁃16 E6 upregulation of DNMT1 through repression of tumor suppressor p53［J］. Oncol Rep, 2010, 24（6）: 1599⁃1604.
［2］	Hanprasertpong J, Tungsinmunkong K, Chichareon S, et al. Correlation of p53 and Ki⁃67 （MIB⁃1） s with clinicopathological features and prognosis of early stage cervical squamous cell carcinomas［J］. J Obstet Gynaecol Res, 2010, 36（3）: 572⁃580. DOI: 10.1111/j.1447⁃0756.2010.01227.x.
［3］	Pan H, Griep AE. Temporally distinct patterns of p53⁃dependent and p53⁃independent apoptosis during mouse lens development［J］. Genes Dev, 1995, 9（17）: 2157⁃2169.
［4］	Sova P, Feng Q, Geiss G, et al. Discovery of novel methylation biomarkers in cervical carcinoma by global demethylation and microarray analysis［J］. Cancer Epidemiol Biomarkers Prev, 2006, 15（1）: 114⁃123. DOI: 10.1158/1055⁃9965.EPI⁃05⁃0323.
［5］	Agrawal N, Dasaradhi PV, Mohmmed A, et al. RNA interference: biology, mechanism, and applications［J］. Microbiol Mol Biol Rev, 2003, 67（4）: 657⁃685.
［6］	Yang HJ. Aberrant DNA methylation in cervical carcinogenesis［J］. Chin J Cancer, 2013, 32（1）: 42⁃48. DOI: 10.5732/cjc.012.10033.
［7］	Lindel K, Rieken S, Daffinger S, et al. The transcriptional regulator gene E2 of the Human papillomavirus （HPV） 16 influences the radiosensitivity of cervical keratinocytes［J］. Radiat Oncol, 2012, 7: 187. DOI: 10.1186/1748⁃717X⁃7⁃187.
［8］	Shin MK, Sage J, Lambert PF. Inactivating all three rb family pocket proteins is insufficient to initiate cervical cancer［J］. Cancer Res, 2012, 72（20）: 5418⁃5427. DOI: 10.1158/0008⁃5472.CAN⁃12⁃2083.
［9］	Vallejo⁃Ruiz V, Velázquez⁃Márquez N, Sánchez⁃Alonso P, et al. Human papillomavirus E7 oncoprotein and its role in the cell transformation［J］. Rev Med Inst Mex Seguro Soc, 2015, 53 Suppl 2: S172⁃177.
［10］	Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer［J］. Nature, 2001, 411（6835）: 342⁃348. DOI: 10.1038/35077213.
［11］	Ting AH, Schuebel KE, Herman JG, et al. Short double⁃stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation［J］. Nat Genet, 2005, 37（8）: 906⁃910. DOI: 10.1038/ng1611.