RNA干涉抑制宫颈癌HPV16E6基因的研究  

Inhibition of HPV16 E6 Oncogene in Cervical Cancer by RNA Interference

作　　者：牛晓宇[1] 彭芝兰[1] 段伟强[2] 王和[1] 陈悦悦[1] 范余娟[1] 程易凡[3] 钱晓蕾[1] 

机构地区：[1]四川大学华西第二医院妇产科,成都610041 [2]四川大学华西医院烧伤整形科 [3]浙江大学附属妇产科医院

出　　处：《四川大学学报（医学版）》2006年第1期14-18,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30371483)资助

摘　　要：目的采用RNA干涉(RNAI)技术干扰宫颈癌HPV16E6基因转录,通过体外和体内实验了解其特异性抑制HPV16E6基因的效率。方法设计合成针对HPV16E6的小干扰RNA(SIRNA),借脂质体转染宫颈癌CASKI细胞,通过测定转染后不同时间点的细胞凋亡率、HPV16E6MRNA及蛋白表达变化了解基因抑制的效率。体内实验中建立子宫颈癌荷瘤裸鼠模型,将E6SIRNA直接注入瘤体,观察肿瘤体积的变化,肿瘤切片HPV16E6免疫组化染色,观察肿瘤坏死和细胞的凋亡。结果HPV16E6SIRNA转染细胞后24H、48H、5D和9D时凋亡率分别为7.7%、11.8%、37.4%和12.6%。RT-PCR显示转染后24H、48H、5D和9D HPV16E6MRNA量减少了77%、83%、59%和41%。WESTERN BLOT显示转染后的HPV16E6蛋白表达在24H、48H、5D明显减少,9D时有所恢复;流式细胞仪HPV16E6蛋白定量测定结果显示,转染后24H、48H、5D和9D蛋白表达抑制率分别为79.7%、80.4%、71.3%和57.4%。体内实验瘤内注射HPV16E6SIRNA明显抑制肿瘤的生长,HPV16E6蛋白表达明显受抑,肿瘤组织坏死和细胞凋亡增加,重复给药较单次给药效果更好。结论体内和体外实验均表明RNA干涉HPV16E6基因的效果具有特异性和高效性。Objective The efficiency of HPV16 E6 gene silenced by RNA interference in vitro and in vivo was assessed. Methods The specific siRNA of HPV16 E6 was designed and transfected into CaSki cells by liposome. Cell apoptotic rates and the changes in HPV16 E6 mRNA and protein before and after transfection were measured. Cervical cancer nude mice models were set up, siRNA was injected directly into subcutaneous tumor. The function of siRNA was evaluated by the changes in tumor volume, HPV16 E6 protein expression and apoptosis of tumor cells. Results In vitro research, the cell apoptotic rates were 7. 7%, 11. 8%, 37. 4% and 12.6% respectively at 24 h, 48 h, 5th day and 9th day after transfection. The HPV16 E6 mRNA was reduced by 77%, 83%, 59% and 41% at 24 h, 48 h, 5th day and 9th day after transfection. The inhibition rates of E6 protein measured by Flow cytometry were 79.7%, 80.4%, 71.3% and 57.4% at 24 h, 48 h, 5th day and 9th day after transfection, which were confirmed by the results of Western blot. In vivo research, E6 siRNA administration groups had great power in inhibiting tumor growth, restraining E6 protein expression, increasing tumor necrosis and apoptosis. The result of repeated injections of siRNA was better than that of single injection. Conclusion RNA interference with HPV16 E6 is specific and highly efficient in vitro and in vivo.

关 键 词：RNA干涉 HPV16 E6 宫颈癌 基因治疗 

分 类 号：R737.33[医药卫生—肿瘤]