Lentivirus-mediated siRNA Targeting Cyclooxygenase-2 Gene Inhibits Human Laryngocarcinoma Cells Proliferation and Invasion_West China Medical Journal

Authors：

WANG Xiaoxia ^1,2 ,  YAO Xiaobao ² , ZHANG Shaoqiang. ¹

1. Department of Otolaryngology, the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China; 2. Department of Otolaryngology, 451 Hospital of PLA, Xi’an, Shaanxi 710054, P. R. China;

Corresponding author：

YAO Xiaobao, Email: xiaoxiawang9139@sina.com.cn

Keywords：

喉癌; 环氧化酶-2基因; 慢病毒; 基因沉默

DOI：

10.7507/1002-0179.20130063

Video：

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目的　构建沉默环氧化酶-2（COX-2）基因重组慢病毒，观察其体外侵袭的抑制作用，从而探讨干扰COX-2抑制喉癌细胞增殖的作用机理，为喉癌的治疗提供新的思路。方法　逆转录聚合酶链反应（RT-PCR）检测COX-2基因在人表皮样喉癌细胞（Hep-2）中的表达情况。利用上海吉凯公司RNA干扰（RNAi）慢病毒表达载体系统，构建针对COX-2基因慢病毒RNAi表达载体。转染Hep-2细胞，干扰COX-2基因的表达，实时定量PCR检测干扰前后基因表达变化。利用生长曲线测定干扰载体转染前后细胞生长速度变化。流式细胞仪检测细胞的生长周期。Boyden侵袭小室法测定体外侵袭力。结果　成功构建了COX-2慢病毒RNAi表达载体，并建立了干扰COX-2基因的Hep-2细胞系。实时定量PCR检测COX-2基因在Hep-2细胞系中过表达被显著抑制。生长曲线测定，COX-2基因干扰后细胞增殖明显变慢。流式细胞仪检测细胞的生长周期可见干扰组诱导Hep-2细胞凋亡，转染G0～G1期细胞数量明显上升，S期细胞减少，表明siRNA干扰Hep-2细胞后，细胞由G0～G1期进入到S期受到阻滞，细胞增殖速度下降。体外侵袭实验中，Hep-2-AS侵袭细胞数（31.0 ± 1.8）显著低于Hep-2细胞（104.0 ± 2.6）及Hep-2-P细胞（99.0 ± 2.7），差异有统计学意义（P＜0.05）。结论　喉癌中过表达的COX-2基因被干扰后表达明显降低并显著抑制细胞的生长速度和侵袭能力。同时验证了COX-2基因RNA干扰在进行抗肿瘤的治疗中潜在的应用前景。

Citation： WANG Xiaoxia,YAO Xiaobao,ZHANG Shaoqiang.. Lentivirus-mediated siRNA Targeting Cyclooxygenase-2 Gene Inhibits Human Laryngocarcinoma Cells Proliferation and Invasion. West China Medical Journal, 2013, 28(2): 195-199. doi: 10.7507/1002-0179.20130063 Copy

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1. 　Das D, Arber N, Jankowski JA. Chemoprevention of colorectal cancer[J]. Digestion, 2007, 76(1): 51-67.
2. 　Wang MT, Honn KV, Nie D. Cyclooxygenases, prostanoids, and tumor progression[J]. Cancer Metastasis Rev, 2007, 26(3-4): 525-534.
3. 　姚小宝, 王晓侠, 张少强, 等. 抑制COX-2表达对逆转喉癌恶性生物学行为的影响[J]. 西安交通大学学报(医学版), 2009, 19(7): 671-673.
4. 　杨健, 胡为民, 任碧轩, 等. RNA干扰体外抑制HBeAg的表达[J]. 细胞与分子免疫学杂志, 2006, 22(5): 670-671, 673.
5. 　Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference[J]. Cancer Cell, 2002, 2(3): 243-247.
6. 　Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase[J]. Science, 1995, 269(5228): 1236-1241.
7. 　Gwyn K, Sinicrope F. Chemoprevention of colorectal cancer[J]. Am J Gastroenterol, 2002, 97(1): 13-21.
8. 　王磊, 陈卫昌, 谢学顺, 等. 塞来昔布对实验性结肠癌原位移植瘤生长及血管形成的影响[J]. 中华肿瘤防治杂志, 2006, 13(17): 1295-1300.
9. 　Chen Z, Zhang X, Li M, et al. Simultaneously targeting epidermal growth factor receptor tyrosine kinase and cyclooxyg-enase-2, an efficient approach to inhibition of squamous cell carcinoma of the head and neck[J]. Clin Cancer Res, 2004, 10(17): 5930-5939.
10. 　王晨, 费小凡. 饶丹, 等. 环氧化酶-2抑制剂致心血管系统的作用机制[J]. 华西医学, 2011, 26(10): 1581-1584.
11. 　Brummelkamp TR, Bernards R. New tools for functional mammalian cancer genetics[J]. Nat Rev Cancer, 2003, 10(10): 781-789.
12. 　Barańska M, Skretkowicz J. Prospects of gene therapy[J]. Wiad Lek, 2007, 60(7-8): 305-311.
13. 　达明绪, 张明鸣, 罗婷, 等. 胃癌COX-2的表达与微血管密度及相关临床病理因素的关系[J]. 华西医学, 2005, 20(1): 12-14.
14. 　宋继荣, 董颖, 潘月娜. 宫颈癌组织中Survivin和Cox-2的表达及相关性[J]. 黑龙江医药科学, 2012, 35(5): 36-38.
15. 　Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors[J]. Cancer Res, 2000, 60(5): 1306-1311.
16. 　Gallo O, Franchi A, Magnelli L, et al. Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis[J]. Neoplasia, 2001, 3(1): 53-61.
17. 　赵晖, 王树成. RNA干扰沉默食管癌COX-2基因的实验研究[J]. 山东大学学报:医学版, 2006, 44(3): 256-259.
18. 　Suzuki O, Abe M. Cell surface N-glycosylation and sialylation regulate galectin-3-induced apoptosis in human diffuse large B cell lymphoma[J]. Oncol Rep, 2008, 19(3): 743-748.
19. 　Takai N, Ueda T, Nishida M, et al. Histone deacetylase inhibitors induce growth inhibition, cell cycle arrest and apoptosis in human choriocarcinoma cells[J]. Int J Mol Med, 2008, 21(1): 109-115.
20. 　Folkman J. Angiogenesis and apoptosis[J]. Cancer Biol, 2003, 13(2): 159-167.

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Lentivirus-mediated siRNA Targeting Cyclooxygenase-2 Gene Inhibits Human Laryngocarcinoma Cells Proliferation and Invasion

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