中华皮肤科杂志

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不同RNA干预技术对人乳头瘤病毒11型E7基因的沉默作用

陈贤祯, 程浩, 汤晓燕, 张行, 朱可建, 叶俊, 林爱华, 岑建萍, 金纳   

  1. 浙江大学医学院附属邵逸夫医院皮肤科, 杭州310016
  • 收稿日期:2006-04-18 出版日期:2007-05-15 发布日期:2007-05-15
  • 通讯作者: 程浩,email:hz_chenghao@126.com E-mail:hz_chenghao@126.com
  • 基金资助:
    国家自然科学基金(30371289)

Silencing of HPV11ET gene expression by different RNA interference technology

CHEN Xian-zhen, CHENG Hao, TANG Xiao-yan, ZHANG Xing, ZHU Ke-jian, YE Jun, LIN Ai-hua, CEN Jian-ping, JIN Na   

  1. Department of Dermatology, Sir Run Shaw Hospital Affiliated to Medical Collage, Zhejiang University, Hangzhou 310016, China
  • Received:2006-04-18 Online:2007-05-15 Published:2007-05-15

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摘要： 目的 探讨RNA干预技术的两种形式在体外对人乳头瘤病毒(HPV)11型E7基因的沉默作用。方法 化学合成1对小片段干扰RNA(siRNA-11E7),同时构建3个短发夹状小RNA(shRNA)表达质粒(pRNAT-11E7 1#、2#、3#),分别转染稳定表达HPV11E7的C57BL/6小鼠黑素瘤细胞系B16细胞株,采用实时荧光定量PCR技术检测细胞转染前后靶基因mRNA的表达水平。结果 siRNA-11E7转染细胞后6、12、24、48、72、96、120 h对靶基因表达的抑制率分别为12.60%、31.41%、41.93%、42.93%、74.35%、32.71%、29.00%;最佳作用浓度为25 nmol/L(抑制率70.06%),最低作用浓度可至3.13 nmol/L(抑制率47.00%)。以0.4μg pRNAT-11E7 1#、2#、3#及上述3个质粒等量混合转染细胞72 h后对靶基因表达的抑制率分别达44.52%、59.26%、89.62%、54.09%,阴性质粒无干预作用。pRNAT-11E7 3#作用6、12、24、48、72、96、120 h对靶基因表达抑制率分别为0、17.50%、40.90%、42.40%、61.90%、51.50%、0%;最佳作用剂量0.2μg。结论 siRNA及shRNA表达载体均可在体外安全高效地沉默HPV11E7基因的表达,其干预作用存在一定的量效性和时效性,且可能存在最佳作用浓度(剂量)和最佳作用时间。

关键词: 基因沉默, RNA干预, 乳头状瘤病毒, 人

Abstract: Objective To investigate the silencing effect of two kinds of RNA interference (RNAi)technology on the expression of HPV11 E7 gene in vitro.Methods A pair of small interference RNA(siRNA)targeting HPV11 E7 gene,named siRNA-11E7,was chemically synthesized,and three short hairpin RNA(shRNA)expression plasmids,i.e.pRNAT-11E7 1#,2#,3#,were constructed.Then a murine melanoma cell line,B16,which stably expressed HPV11 E7 gene,was transfected respectively with the siRNA-11E7 and pRNAT-11E7 1#,2#,3#,etc.The level of E7 mRNA expression was detected in these cells by real-time fluorescent quantitative PCR before and after the transfection.Results siRNA-11E7 could inhibit the E7 gene mRNA expression in B16 cells by 12.60%,31.41%,41.93%,42.93%,74.35%,32.71% and 29.00% at 6 h,12 h,24 h,48 h,72 h,96 h and 120 h after the transfection,respectively.The optimal dose of siRNA was 25 nmol/L with an inhibition rate of 70.06%,and the lowest effective dose was 3.13 nmol/L with an inhibition rate of 47%.The 0.4μg of pRNAT-11E7 1#,2#,3# and a mix of the 3 plasmids could decrease the target gene expression by 44.52%,59.26%,89.62%,54.09%,respectively,while the negative plasmid had no inhibitory effect on the gene expression,pRNAT-11E7 3# could decrease the E7 gene expres-sion by 0,17.50%,40.90%,42.40%,61.90%,51.50% and 0,at 6 h,12 h,24 h,48 h,72 h,96 h and 120 h after the transfection,respectively.The optimal dose ofpRNAT-11E7 was 0.2μg.Conclusions Both siRNA and shRNA expression plasmids could efficiently and safely silence the HPV11E7 mRNA expression in a time-and dose-dependent manner,and the optimal action dose and time is likely to be identified.

Key words: Gene silencing, RNA interference, Papillomavirus,human