HPV16/18 E6/E7基因沉默对SiHa/HeLa细胞生物学行为影响的研究
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摘要
目的高危型人乳头瘤病毒(high risk human papillomavirus，HR-HPV)的E6/E7是引起宫颈癌的主要致癌基因，在宫颈癌筛查与HPV治疗中具有重大的临床意义，目前E6/E7在宫颈癌致癌中机制尚不十分明确。本研究目的在于通过观察E6/E7基因沉默对于宫颈癌细胞株生物学行为的影响，包括体外的增殖、凋亡、侵袭和裸鼠体内成瘤能力，同时观察E6/E7沉默后宫颈癌细胞对抗肿瘤药物顺铂耐受性的改变以及细胞内hTERC基因表达的变化，探讨HPV E6/E7在宫颈癌发生发展过程中的作用，并推测其发挥作用可能的分子机制，初步评价shRNA干扰对于HPV感染和抗肿瘤治疗领域的应用价值。
方法1.shRNA质粒构建针对HPV16与HPV18E6/E7mRNA序列，分别设计3对shRNA特异性序列，构建shRNA载体共6对，利用电击转染法转染HPV16整合的SiHa细胞株与HPV18整合的HeLa细胞株，筛选出稳定转染的单细胞克隆细胞株（pSi-16-NC, pSi-16-1, pSi-16-2, pSi-16-3与pSi-18-NC, pSi-18-1, pSi-18-2, pSi-18-3）。
2.体外研究：①应用RT-PCR检测转染后24h、48h、72h，HPV16与HPV18E6与E7mRNA表达；②应用实时定量RT-PCR检测稳定转染细胞株中HPV16与HPV18E6与E7mRNA表达；③应用Western-blot检测转染后SiHa细胞和HeLa细胞中p53、pRb和p16抑癌蛋白的表达；④应用MTT测定转染后SiHa细胞和HeLa细胞的生长增殖能力；Transwell实验检测细胞迁移能力；流式细胞技术检测shRNA转染后SiHa细胞和HeLa细胞的周期分布；⑤顺铂作用48h后流式细胞技术检测细胞总凋亡与早、晚期凋亡；⑥应用FISH检测shRNA稳定转染后细胞内hTERC基因表达变化。
3.体内研究：应用SiHa细胞和HeLa细胞稳定转染细胞株分别建立裸鼠人宫颈癌模型（2×10~6个细胞/只）。①通过比较肿瘤体积和重量，观察E6/E7沉默对裸鼠肿瘤生长的抑制作用；②应用FISH检测shRNA转染后癌组织中hTERC基因表达变化。
结果1. shRNA质粒的干扰作用：针对E6/E7设计的6组pSi-shRNA载体能够有效地抑制HPV16E6/E7mRNA与HPV18E6/E7mRNA的表达。抑制效果因选择的靶点位置不同而存在差异。在SiHa细胞中，pSi-16-1, pSi-16-2, pSi-16-3对E6和E7的抑制率分别为85.4%与54.9%；91.9%与63.2%；84.7%与71.1%。在HeLa细胞中，pSi-18-1, pSi-18-2, pSi-18-3对E6和E7的抑制率分别为91.3%与55.8%；90.4%与7.8%；87.2%与81.0%。
2.体外研究：①q RT-PCR结果显示，pSi-shRNA质粒能够有效沉默HPV16/18E6与E7mRNA的表达，见结果一；②转染前后SiHa细胞和HeLa细胞中p53、pRb和p16抑癌蛋白的表达均有上调；③M TT试验结果显示，E6/E7沉默使SiHa细胞和HeLa细胞的增殖活性降低，部分组（pSi-16-2, pSi-16-3, pSi-18-3）与对照组比较有显著性差异（P＜0.05）；④Transwell实验结果显示，E6/E7沉默使肿瘤细胞迁移能力下降,部分组（pSi-16-2, pSi-16-3, pSi-18-1, pSi-18-2, pSi-18-3）与对照组比较有显著性差异（P＜0.05）；⑤细胞周期检测结果表明E6/E7基因沉默后，G0/G1期细胞比例增加，S期细胞比例降低；⑥检测顺铂作用48小时后细胞凋亡情况显示，转染后细胞总调亡率增加，表明E6/E7沉默使细胞对顺铂的敏感性增加；⑦h TERC基因检测发现E6/E7基因沉默使hTERC表达下调，正常细胞比例增加。
3.体内研究：①E6/E7沉默能抑制宫颈癌细胞的在裸鼠体内的生长，抑制裸鼠肿瘤的体积，与对照组比较有显著性差异（P＜0.01）；②E6/E7抑制降低了裸鼠体内肿瘤细胞hTERC基因的表达，使hTERC基因正常表达的细胞比例增加。
结论重组质粒pSi-shRNA能够使E6/E7在宫颈癌细胞株SiHa与HeLa细胞中的表达下调，其抑制率可达到60%~90%。E6/E7基因转录水平的沉默使宫颈癌细胞SiHa与HeLa细胞中抑癌蛋白p53、pRb、p16的表达增加，细胞生长受抑制，迁移能力降低，同时使处于G0/G1期细胞比例增加，S期细胞比例减少。综上表明E6/E7基因在宫颈癌细胞生长、增殖、迁移以及细胞周期调控等方面起着重要的作用，与肿瘤细胞的恶性生物学行为密切相关。顺铂药物实验表明E6/E7的抑制使宫颈癌细胞对顺铂敏感性增加，促进了细胞的凋亡。FISH技术检测E6/E7稳定抑制的细胞中hTERC基因的表达，发现E6/E7表达与hTERC基因扩增具有相关性。
Objective: Human papillomavirus (HPV) oncogenes E6and E7play an important role incervical cancer. The E6/E7oncogenes show an important value in the cervical cancerscreening and treatment of HPV. However, the carcinogenic mechanism of E6/E7remainsunclear. In this study, we observed the malignant biological behavior in stable-transfectedcervical cancer cells, which include the expression of tumor suppressor proteins p53, pRband p16, cell proliferation, cell cycles, migration. In addition, we detected the cellapoptosis after treatment of Cisplatin and the amplification of hTERC gene. We aimed toexplore the role of E6/E7in the carcinogenesis and progression of cervical cancer and tosuppose molecular mechanism, and to assess the value of shRNA in HPV treatment andanti-tumor.
Methods:1. shRNA plasmid construction: we designed6shRNA consequences accordingthe designed rules aimed at HPV16and18E6/E7mRNA respectively. Then plasmids weretransfected into SiHa and HeLa cells by Electrical-transfection. Stable-transfected silencedmomclonal cells expressing shRNA and negative control shRNA were selected for furtherexperiments.
2. In vitro:①Using reverse-transcription PCR, the effects of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;②By real-time quantitative RT-PCR, theeffect of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;③ByWestern-blot, the expression of anti-tumor proteins p53, pRb, and p16in SiHa cell andHeLa cell were investigated;④The growth rate of HeLa cell and SiHa cell treated withshRNA was determined with MTT assay; Transwell assay be used to observe cellmigration in vitro; Cell cycles and apoptosis of SiHa cell HeLa cell after48h treated withcisplatin were observed with flow cytometer;⑤Amplication of hTERC gene wasdetected by FISH.
3. In vivo: Nude mices were injected2×10~6the stable-transfected SiHa cell and HeLa cells, respectively.①Volumn of transplantation tumors were measured and compared;②Expression of hTERC gene on tumor tissue was detected by FISH.
Results:1. Interference of shRNA: we successfully constructed6shRNA plasmids. Thesequence-specifical shRNA suppressed the HPV16/18E6/E7mRNA expression effectively.In SiHa cells, quantitative real-time PCR showed that E6and E7expression reduced byshRNA pSi-16-1, pSi-16-2, pSi-16-3were85.4%and54.9%；91.9%and63.2%；84.7%and71.1%, respectively. And in HeLa cells, the suppression ratios were91.3%and55.8%；90.4%and7.8%；87.2%and81.0%, respectively.2.In vitro:①pSi-shRNA can silence expression of HPV E6/E7mRNA effectively;②pSi-shRNA can increase the expression of p53, pRb and p16in SiHa cell and HeLa cell;③It was confirmed by MTT assay that pSi-shRNA can significantly inhibit theproliferation of SiHa cell and HeLa cell（P＜0.05);④After be treated by colchicine, thepropotions of cell in G0/G1stage increased. However, the propotions of cell in S stagedecreased;⑤By Transwell assay, cell migration ability were weaken;⑥After betreated by cisplatin for48h, apotosis rate were increase. Silencing of E6/E7increased SiHacell sensitivity to cisplatin;⑦Silencing of E6/E7inhibited expression of hTERC gene.The normal cell was increased and the cell signal of4:4was decreased.3. In vivo:①the growth of tumor was inhibited by silencing of E6/E7, the volume of thetumor in pSi-shRNA were lower than that of negative control (P＜0.01）;②the expressionof hTERC gene were dow-regulation by silencing of E6/E7.
Conclusion: The recombinant plasmids can silence the expression of HPV16and HPV18E6/E7successfully. Silencing of HPV E6/E7resulted in the expression of p53、pRb、p16increase, and cell growth inhibition, migration ability weaken, and made the cell cycleasset in G0/G1stage in SiHa and HeLa cells. In addition, Silencing of HPV E6/E7increasedcell sensitivity to cisplatin. A closely relationship between the E6/E7and hTERC gene inSiHa and HeLa both in vitro and in vivo was found.
方法1.shRNA质粒构建针对HPV16与HPV18E6/E7mRNA序列，分别设计3对shRNA特异性序列，构建shRNA载体共6对，利用电击转染法转染HPV16整合的SiHa细胞株与HPV18整合的HeLa细胞株，筛选出稳定转染的单细胞克隆细胞株（pSi-16-NC, pSi-16-1, pSi-16-2, pSi-16-3与pSi-18-NC, pSi-18-1, pSi-18-2, pSi-18-3）。
2.体外研究：①应用RT-PCR检测转染后24h、48h、72h，HPV16与HPV18E6与E7mRNA表达；②应用实时定量RT-PCR检测稳定转染细胞株中HPV16与HPV18E6与E7mRNA表达；③应用Western-blot检测转染后SiHa细胞和HeLa细胞中p53、pRb和p16抑癌蛋白的表达；④应用MTT测定转染后SiHa细胞和HeLa细胞的生长增殖能力；Transwell实验检测细胞迁移能力；流式细胞技术检测shRNA转染后SiHa细胞和HeLa细胞的周期分布；⑤顺铂作用48h后流式细胞技术检测细胞总凋亡与早、晚期凋亡；⑥应用FISH检测shRNA稳定转染后细胞内hTERC基因表达变化。
3.体内研究：应用SiHa细胞和HeLa细胞稳定转染细胞株分别建立裸鼠人宫颈癌模型（2×10~6个细胞/只）。①通过比较肿瘤体积和重量，观察E6/E7沉默对裸鼠肿瘤生长的抑制作用；②应用FISH检测shRNA转染后癌组织中hTERC基因表达变化。
结果1. shRNA质粒的干扰作用：针对E6/E7设计的6组pSi-shRNA载体能够有效地抑制HPV16E6/E7mRNA与HPV18E6/E7mRNA的表达。抑制效果因选择的靶点位置不同而存在差异。在SiHa细胞中，pSi-16-1, pSi-16-2, pSi-16-3对E6和E7的抑制率分别为85.4%与54.9%；91.9%与63.2%；84.7%与71.1%。在HeLa细胞中，pSi-18-1, pSi-18-2, pSi-18-3对E6和E7的抑制率分别为91.3%与55.8%；90.4%与7.8%；87.2%与81.0%。
2.体外研究：①q RT-PCR结果显示，pSi-shRNA质粒能够有效沉默HPV16/18E6与E7mRNA的表达，见结果一；②转染前后SiHa细胞和HeLa细胞中p53、pRb和p16抑癌蛋白的表达均有上调；③M TT试验结果显示，E6/E7沉默使SiHa细胞和HeLa细胞的增殖活性降低，部分组（pSi-16-2, pSi-16-3, pSi-18-3）与对照组比较有显著性差异（P＜0.05）；④Transwell实验结果显示，E6/E7沉默使肿瘤细胞迁移能力下降,部分组（pSi-16-2, pSi-16-3, pSi-18-1, pSi-18-2, pSi-18-3）与对照组比较有显著性差异（P＜0.05）；⑤细胞周期检测结果表明E6/E7基因沉默后，G0/G1期细胞比例增加，S期细胞比例降低；⑥检测顺铂作用48小时后细胞凋亡情况显示，转染后细胞总调亡率增加，表明E6/E7沉默使细胞对顺铂的敏感性增加；⑦h TERC基因检测发现E6/E7基因沉默使hTERC表达下调，正常细胞比例增加。
3.体内研究：①E6/E7沉默能抑制宫颈癌细胞的在裸鼠体内的生长，抑制裸鼠肿瘤的体积，与对照组比较有显著性差异（P＜0.01）；②E6/E7抑制降低了裸鼠体内肿瘤细胞hTERC基因的表达，使hTERC基因正常表达的细胞比例增加。
结论重组质粒pSi-shRNA能够使E6/E7在宫颈癌细胞株SiHa与HeLa细胞中的表达下调，其抑制率可达到60%~90%。E6/E7基因转录水平的沉默使宫颈癌细胞SiHa与HeLa细胞中抑癌蛋白p53、pRb、p16的表达增加，细胞生长受抑制，迁移能力降低，同时使处于G0/G1期细胞比例增加，S期细胞比例减少。综上表明E6/E7基因在宫颈癌细胞生长、增殖、迁移以及细胞周期调控等方面起着重要的作用，与肿瘤细胞的恶性生物学行为密切相关。顺铂药物实验表明E6/E7的抑制使宫颈癌细胞对顺铂敏感性增加，促进了细胞的凋亡。FISH技术检测E6/E7稳定抑制的细胞中hTERC基因的表达，发现E6/E7表达与hTERC基因扩增具有相关性。
Objective: Human papillomavirus (HPV) oncogenes E6and E7play an important role incervical cancer. The E6/E7oncogenes show an important value in the cervical cancerscreening and treatment of HPV. However, the carcinogenic mechanism of E6/E7remainsunclear. In this study, we observed the malignant biological behavior in stable-transfectedcervical cancer cells, which include the expression of tumor suppressor proteins p53, pRband p16, cell proliferation, cell cycles, migration. In addition, we detected the cellapoptosis after treatment of Cisplatin and the amplification of hTERC gene. We aimed toexplore the role of E6/E7in the carcinogenesis and progression of cervical cancer and tosuppose molecular mechanism, and to assess the value of shRNA in HPV treatment andanti-tumor.
Methods:1. shRNA plasmid construction: we designed6shRNA consequences accordingthe designed rules aimed at HPV16and18E6/E7mRNA respectively. Then plasmids weretransfected into SiHa and HeLa cells by Electrical-transfection. Stable-transfected silencedmomclonal cells expressing shRNA and negative control shRNA were selected for furtherexperiments.
2. In vitro:①Using reverse-transcription PCR, the effects of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;②By real-time quantitative RT-PCR, theeffect of shRNA on HPV16E6/E7and HPV18E6/E7mRNA was studied;③ByWestern-blot, the expression of anti-tumor proteins p53, pRb, and p16in SiHa cell andHeLa cell were investigated;④The growth rate of HeLa cell and SiHa cell treated withshRNA was determined with MTT assay; Transwell assay be used to observe cellmigration in vitro; Cell cycles and apoptosis of SiHa cell HeLa cell after48h treated withcisplatin were observed with flow cytometer;⑤Amplication of hTERC gene wasdetected by FISH.
3. In vivo: Nude mices were injected2×10~6the stable-transfected SiHa cell and HeLa cells, respectively.①Volumn of transplantation tumors were measured and compared;②Expression of hTERC gene on tumor tissue was detected by FISH.
Results:1. Interference of shRNA: we successfully constructed6shRNA plasmids. Thesequence-specifical shRNA suppressed the HPV16/18E6/E7mRNA expression effectively.In SiHa cells, quantitative real-time PCR showed that E6and E7expression reduced byshRNA pSi-16-1, pSi-16-2, pSi-16-3were85.4%and54.9%；91.9%and63.2%；84.7%and71.1%, respectively. And in HeLa cells, the suppression ratios were91.3%and55.8%；90.4%and7.8%；87.2%and81.0%, respectively.2.In vitro:①pSi-shRNA can silence expression of HPV E6/E7mRNA effectively;②pSi-shRNA can increase the expression of p53, pRb and p16in SiHa cell and HeLa cell;③It was confirmed by MTT assay that pSi-shRNA can significantly inhibit theproliferation of SiHa cell and HeLa cell（P＜0.05);④After be treated by colchicine, thepropotions of cell in G0/G1stage increased. However, the propotions of cell in S stagedecreased;⑤By Transwell assay, cell migration ability were weaken;⑥After betreated by cisplatin for48h, apotosis rate were increase. Silencing of E6/E7increased SiHacell sensitivity to cisplatin;⑦Silencing of E6/E7inhibited expression of hTERC gene.The normal cell was increased and the cell signal of4:4was decreased.3. In vivo:①the growth of tumor was inhibited by silencing of E6/E7, the volume of thetumor in pSi-shRNA were lower than that of negative control (P＜0.01）;②the expressionof hTERC gene were dow-regulation by silencing of E6/E7.
Conclusion: The recombinant plasmids can silence the expression of HPV16and HPV18E6/E7successfully. Silencing of HPV E6/E7resulted in the expression of p53、pRb、p16increase, and cell growth inhibition, migration ability weaken, and made the cell cycleasset in G0/G1stage in SiHa and HeLa cells. In addition, Silencing of HPV E6/E7increasedcell sensitivity to cisplatin. A closely relationship between the E6/E7and hTERC gene inSiHa and HeLa both in vitro and in vivo was found.
引文
[1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics [J].CA Cancer J Clin,2011,61(2):69-90.
[2] Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer[J]. Clin Sci (Lond),2006,110(5):525-541.
[3] Tavassoli FA, Davilee P. WHO classification of tumor. Pathology&genetics tumors ofthe breast and female genital organs[M] Lion: ARC Press,2003:266.
[4] Cox JT. Human papillomavirus testing in primary cervical screening and abnormalPapanicolaou management [J]. Obstet Gynecol Surv,2006,61(6): S15-25.
[5] Wei Wang, Ni Sima, Debo Kong,et al. Selective targeting of HPV-16E6/E7in cervicalcancer cells with a potent oncolytic adenovirus and its enhanced effect withradiotherapy in vitro and vivo[J].Cancer Letters,2010,291(1):67-75.
[6] Hong D, Lu W, Ye F, et al. Gene silencing of HPV16E6/E7induced bypromoter-targeting siRNA in SiHa cells, Br J Cancer,2009,101(10):1798-1804.
[7] Murphy N, Ring M, Killalea AG, et al. p16INK4A as a marker for cervicaldyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears [J]. J ClinPathol,2003,56(1):56-63.
[8] Klaes R, Benner A, Friedrich T, et al. p16INK4a immunchistochemis-try inprovesinterobserver agement in the diagnosis of cervical inteaepthelial neoplasis [J]. Am JSurg Pathol,2002,26(11):1389-1399.
[9] Mutirangura A, Sriuranpong V, Termrunggraunglert W, et al. Telomerase activity andhuman papillomavirus in malignant, premalignant and benign cervical lesions [J]. Br JCancer,1998,78(7):933-939.
[10] Kailash U, Soundararajan CC, Lakshmy R, et al. Telomerase activity as an adjunct tohigh-risk human papillomavirus types16and18and cytology screening in cervicalcancer. Br J Cancer,2006,95(9):1250-7.
[11] Tu Z, Zhang AP, Wu RF, et al. Genomic amplification of the human telomeraseRNA gene for differential diadnosis of cervical disorder [J]. Cancer Genet Cytogen,2009,191(1):10-16.
[12] Gonzalez VM, Fuertes MA, Alonso C, et al. Is cisplatin-induced cell death alwaysproduced by apoptosis?[J]Mol Pharmacol,2001,59(4):657-663.
[13] Losa JH, Parada Cobo C, Viniegra JG, et al.Role of the p38MAPK pathway incisplatinbased therapy [J]. Oncogene2003,22(26):3998-4006.
[1] Tuschl T, Borkhardt A. Small interfering RNAs:a revolutionary tool for the analysis ofgene function and gene therapy[J]. Mol Interv,2002;2(3):158-67.
[2] Boutla A, Delidakis C, Livadaras I, et al. Variations of the3’protruding ends insynthetic short interfering RNA(siRNA) tested by microinjection in Drosophilaembryos [J]. Oligonucleotides,2003;13(5):295-301.
[3] Balsitis S, Dick F, Lee D, et al. Examination of the pRb-dependent andpRb-independent functions of E7in vivo.J Virol,2005,79(17):11392-402.
[4] Shai A, Brake T, Somoza C, Lambert PF. The human papillomavirus E6oncogenedysregulates the cell cycle and contributes to cervical carcinogenesis through twoindependent activities. Cancer Res,2007,67(4):1626-35.
[5] Elbashir SM, Harborth J, Lendckel W, et al. Duplexes of21-nucleotide RNAs mediateRNA interference in cultured mammalian cells [J]. Nature,2001;4119(6836):494-498.
[6] Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs formediating efficient RNAi in Drosophila melanogaster embryolysate [J]. EMBO J,2001,20(23):6877-88.
[7] Peter MW, Ming-Bo W, Tony L. Gene silencing as an adaptive defence against viruses[J]. Nature,2001,411(14):8342842.
[8] van Rij RP, Andino R. The silent treatment: RNAi as a defense against virus infectionin mammals [J]. Trends Biotechnol,2006,24(4):186-93.
[9] Schütz S, Sarnow P. Interaction of viruses with the mammalian RNA interferencepathway [J]. Virology,2006,5;344(1):151-7.
[10] Masiero M,Nardo G, Indraccolo S. RNA interference: implications for cancertreatment [J]. Mol Aspects Med,2007,28(1):143-166.
[11] Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible andgenome-wide screens [J]. Drug Discov Today,2006,11(21-22):975-82.
[12] Jiang M, Milner J. Selective Silencing of Viral Gene E6and E7Expression inHPV-Positive Human Cervical Carcinoma Cells Using Small Interfering RNAs [J].Methods Mol Biol,2004,292:401-20.
[13] Cho WC, Haryoung P, Young SC, et al. HPV E6antisense induces apoptosis in CaSkicells via suppression of E6splicing [J]. Exp Mol Med,2002,34(2):159-166.
[14] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells [J]. Science,2002,19;296(5567):550-3.
[15] Meissner JD. Nucleotide sequences and further characterization of humanpapillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma celllines [J]. Gen Virol,1999,80(pt7):1725-33.
[1] DiPaolo JA, Alvarez-Salas LM. Advances in the development of therapeutic nucleicacids against cervical cancer [J]. Expert Opin Biol Ther,2004,4(8):1251-57.
[2] Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application[J].Nat Rev Cancer,2002,2(5):342-50.
[3] Butz K, Ristriani T, Hengstermann A, et al. siRNA targeting of the viral E6oncogeneefficiently kills human papillomavirus-positive cancer cells [J]. Oncogene,2003,22(38):5938-45.
[4] Madrigal M, Janicek MF,Sevin B,et al.Averette HE.In vitro antigene therapy targetingHPV-16E6and E7in cervical carcinoma.Gynecologic [J]. Oncology.1997,61:18-25.
[5] Jiang M, Milner J. Selective Silencing of Viral Gene E6and E7Expression inHPV-Positive Human Cervical Carcinoma Cells Using Small Interfering RNAs [J].Methods Mol Biol.2004,292:401-20.
[6]吴剑，藤国英.中药诱导肿瘤细胞凋亡的研究进展［J］.国际医药卫生导报，2007,13(8):114-117．
[7] Lee KH, Yim EK, Kim CJ, et al. Proteomic analysis of anticancer effects by paclitaxeltreatment in cervical cancer cells[J]. Gynecol Oncol，2005，98(1):45-53．
[8] Forsmo S，Buhaug H，Skjeldestad FE，et al. Treatment of pre-invasive conditionsduring opportunistic screening and its effectiveness on cervical cancer incidence inone Norwegian county[J]. Int J Cancer,1997,71(1):4-8．
[9]豆长明,李继承.天花粉蛋白对HepA-H细胞和Hela细胞抑癌活性研究[J].中国病理生理杂志,2005,21(5):980-984．
[10]齐玉环,吴丹.细胞凋亡和p53在宫颈癌及宫颈上皮内瘤变中的表达[J].现代妇产科学进展,2009,9(1):16-18．
[11] Wang X, Martindale JL, Holbrook NJ. Requirement for ERK activation incisplatin-induced apoptosis [J]. J Biol Chem,2000,275(50):39435-39443.
[12]廖英俊，汤浩，金亚平.抗癌药顺铂对小鼠的耳、肾和肝毒性及其机制的研究[J].中国药理学通报,2004,(1):82-85.
[13]丁大连.顺铂及其耳毒性[J].中华耳科学杂志,2008,6(2）:125-133.
[14]柳萌,杨迪生.顺铂耐药机制的研究进展[J].现代中西医结合杂志，2007，16（6）:856-858.
[15]陈道桢,耿金花,薛文群,等.染色体不稳定性及其与妇科肿瘤形成关系的研究进展[J].东南大学学报（医学版），2004,23:355-357．
[16] Umayahara K, Numa F, Suehiro Y, et al. Comparative genomic hybridization detectsgenetic alteration during early stages of cervical cancer progression [J]. GenesChromosomes Cancer,2002,33:98-102.
[17] Kloth JN, Osting J, van Wezel T, et al. Combined arraycomparative genomichybridization and single-nucleotidepolymorphism-loss of heterozygosity analysisreveals complex genetic alterations in cervical cancer[J]. BMC Genomic,2007,8(20):53-66.
[18] Kerstin HH, Viktor J, Philip E, et al. Detection of genomic amplification of the humantelomerase gene (hTERC) in cytologic specimens as a genetic test for the diagnosis ofcervical dysplasia[J]. American Journal of Pathology,2003,163(4):1406-1416.
[1] Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential astherapeutics [J]. Nat Rev Drug Discov,2004,3(4):318-329.
[2] Lundstrom K. Prospects of treating neurological diseases by gene therapy[J]. Curr OpinInvestig Drugs,2007,8(1):34-40.
[3]廖维甲,梅铭惠,徐静,等.小干扰RNA抑制肝癌细胞株BEL7402ICAM-1基因表达的研究[J].中国现代医学杂志,2006,16(20):3065-3068.
[4] Yamada Y, Hamajima N, Kato T, et al. Association of a polymorphism of thephospholipase D2gene with the prevalence of colorectal cancer[J].J Mol Med,2003;81(2):126-131.
[5] Chen JQ, Zhan WH, He YL, et al. Expression of heparanase gene, CD44v6, MMP-7and nm23protein and their relationship with the invasion and metastasis of gastriccarcinomas[J].World J Gastroenterol,2004;10(6):776-782.
[6] Mcmanus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J].Nat.Rev Genet,2002;3(10):737-747.
[1] Andraw issm. Cervical cancer vaccines available in2007[J].Drug Discov Today,2005,10(14):949-950.
[2] Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):71-96.
[3] Bulk S,Berkhof J,Bulkmans NW,et al.Preferential risk of HPV16for squamous cellcarcinoma and of HPV18for adenocarcinma of the cervix compared to woman withnormal normal cytology in the Netherlans[J] Br J Cancer,2006,94(1):171-175.
[4] Clifford GM, Gallus S, Herrero R, et al. Worldwide distribution of humanpapillomavirus types in cytologically normal woman in the International Agency forResearch on Cancer HPV prevalence surveys: a pooled analysis[J]. Lancet,2005;366(9490):991-998.
[5] Woodman CB, Collins S, Winter H, et al. Natural history of cervical humanpapillomavirus infection in young women: a longitudinal cohorts study[J]. Lancet,2001,357(9271):1831-1836.
[6] Plummer M, Schiffman M, Castle PE, et al. A2-year prospective study of humanpapillomavirus persistence among women with a cytological diagnosis of atypicalsquamous cells of undetermined significance or low-grade squamous intraepitheciallesion[J]. J Infect Dis,2007,195(11):1560-1562.
[7] Bulkmans NW, Berkhof J, Bulk S, et al. High-risk HPV type-specific clearance rates inCervical screening[J]. Br J Cancer,2007,96(9):1419-1424.
[8] Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer[J]. Clin Sci (Lond),2006,110(5):525-541.
[9] Tavassoli FA, Davilee P. WHO classification of tumor. Pathology&genetics tumors ofthe breast and female genital organs [M]. Lion: ARC Press,2003,266.
[10] Cox JT. Human papillomavirus testing in primary cervical screening and abnormalpapanicolaou management[J]. Obstet Gynecol Surv,2006,61(6):15-25.
[11] Siivia DS, Mireia D, Xavier C, et al. Worldwide prevalence and genotype distributionof cervical human papillomavirus DNA in women with normal cytology: ameta-analysis[J]. Lancet Infect Dis,2007,7(7):453-459.
[12] Castellsague X, Diaz M, Sanjose S, et al. Worldwide human papillomavirus etiologyof cervical adenocarcinoma and its cofactors: implication for screening andprevention [J]. Natl Cancer Inst,2006,98(5):303-315.
[13] Park JS, Ryu HS, Chang SJ, et al. The association of the cervical intraepithelialneoplasia and Human papillomavirus viral Load[J]. Korean J Obstet Gynecol,2005；48（28）：88-95.
[14] Yong MK，Jim YP，Kyung ML，et al. Does pretreatment HPV viral load correlate withprognosis in patients with early stage cervical carcinoma [J]. Gynecol Oncol,2008,19(2):113-116.
[15] Cheung JL, Cheung TH, Ng CW, et al. Analysis of human papillomavirus type18load and integration status from low-grade cervical lesion to invasive cervical cancer[J]. Journal of Clinical Microbiology,2009;47(2):287-293.
[16] Dalstein V, Riethmuller D, Prétet JL, et al. Persistence and load of high-risk HPV arepredictors for development of high-grade cervical lesions: A longitudinal Frenchcohort study[J]. Int J Cancer,2003,106(3):396-403.
[17] Akhtar M，Tu JJ.Human papilloma virus (HPV) vaccines：a major advance againstcervical cancer[J]. Adv Anat Pathol,2006,13(6):336-337.
[18] Villa LL, Costa RL, Petta CA, et al. Prophylactic quadrivalent human papillomavirus(types6,11,16,and18) L1virus-like particle vaccine in young women: a randomiseddouble-blind placebo-controlled multi-centre phase Ⅱ efficacy trial[J]. Lancet Oncol,2005,6(5):271-278.
[19] Harper DM, Franco EL, Wheeler CM, et al. Sustained efficacy up to4.5years of abivalent L1virus-like particle vaccine against human papillomavirus types16and18:follow-up from a randomized control trial[J]. Lancet,2006,367(9518):1247-1255.
[1] Garnett GP, Kim JJ，French K，et a1．Chapter21modelling the impactof HPVvaccinesion cervical cancer and screening programmes [J]. Vaccine.2006，24[Suppl3]：178-186．
[2] Cole ST, Danos O. Nucleotide sequence and comparative analysis of the humanpapillomavirus type18genome. Phylogeny of papillomaviruses and repeatedstructure of the E6and E7gene products [J]. J Mol Biol.1987,193(4),599–608.
[3] Keegan H, Mc Inerney J, Pilkington L, et al. Comparison of HPV detectiontechnologies: Hybrid capture2, PreTect HPV-Proofer and analysis of HPV DNAviral load in HPV16, HPV18and HPV33E6/E7mRNA positive specimens[J]. JVirol Methods.2009,155(1):61-66.
[4] Molden T, Kraus I, Skomedal H, et al. PreTect HPV-Proofer: real-time detection andtyping of E6/E7mRNA from carcinogenic human papillomaviruses[J]. J VirolMethods.2007,142(1-2):204-212.
[5] Zanier K, Charbonnier S, Baltzinger M, et al. Kinetic analysis of the interactions ofhuman papillomavirus E6oncoproteins with the ubiquitin ligase E6AP usingsurface plasmon resonance[J].J Mol Biol,2005,349(2):401-412.
[6] Massimi P, Shai A, Lambert P, et al. HPV E6degradation of p53and PDZcontaining substrates in an E6AP null background [J].Oncogene,2008,27(12):1800-1804.
[7] Camus S, Higgins M, Lane DP, et al. Differences in the ubiquitination of p53byMdm2and the HPV protein E6[J]. FEBS Lett.2003;536(1-3):220-224.
[8] Türkcüoglu I， Tezcan S，Kaygusuz G，et al. The role of p53，Bcl-2and Ki-67inpremalignant cervical lesions and cervical cancer［J. Eur J Gynaecol Oncol，2007，28（4）:290-293.
[9] Looi ML，Dali AZ，Ali SA，et al. Expression of p53，bcl-2and Ki-67in cervicalintraepithelial neoplasia and invasive squamous cellcarcinoma of the uterine cervix［J］. Anal Quant Cytol Histol.2008,30（2）:63-70.
[10] Liang CW, Lin MC, Hsiao CH，et al. Papillary squamous intraepithelial lesions ofthe uterine cervix: human papillomavirus-dependent changes in cell cycleexpression and cytologic features[J]. Hum Pathol.2010,41(3):326-335.
[11] Kruse AJ，Skaland I，Munk AC，et al. Low p53and retinoblastoma proteinexpression in cervical intraepithelial neoplasia grade3lesions is associated withcoexistent adenocarcinoma in situ［J］. Hum Pathol.2008，39（4）:573-578.
[12] Liu X， Clements A, Zhao K， etal. Structure of the human Papillomavirus E7oncoprotein and its mechanism for inactivation of the retinoblastoma tumorsuppressor[J]. J Biol Chem.2006,281(1)：578–586.
[13] Fiedler M, Müller-Holzner E, Viertler HP，et al. High level HPV-16E7oncoproteinexpression correlates with reduced pRb-levels in cervical biopsies.[J]. FASEB J.2004,18(10):1120-1122.
[14] Balsitis S, Dick F, Dyson N, Lambert PF. Critical roles for non-pRb targets ofhuman papillomavirus type16E7in cervical carcinogenesis[J]. Cancer Res.2006,66(19):9393-9400.
[15] Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16IN K4A as an indicatorfor human papillomavirus oncogenic activity in cervical squamous neoplasia [J].Int J Gynecol Cancer.2006,16(l):347-353.
[16] Kalof AN, Evans MF, Simmons AL, et al. p16IN K4A immuno-expression andHPV in situ hybridization signal patterns: Potential markers of high-grade cervicalintraepithelial neoplasia [J]. Am J Surg Pathol.2005,29(5):674-679.
[17] Kanao H, Enomoto T, Ueda Y, et a l. Correlation betw een pl4/p16IN K4Aexpression and HPV infection in uterine cervical cancer [J]. Cancer Lett.2004,213(1):31-37.
[18］Longatto FA，Etlinger D，Pereira SM，et al. The association of p16INK4A andfragile histidine triad gene expression and cervical lesions[J]. J Low Genit Tract Dis.2007，11（3）:151-157.
[19］Redman R, Rufforny I，Liu C，et al. The utility of p16（Ink4a） in discriminatingbetween cervical intraepithelial neoplasia1and nonneoplastic equivocal lesions ofthe cervix [J]. Arch Pathol Lab Med.2008,132（5）:795-799.
[20］Kong CS， Balzer BL， Troxell ML, et al. p16INK4A immunohistochemistry issuperior to HPV in situ hybridization for the detection of high-risk HPV in atypicalsquamous metaplasia [J]. Am J Surg Pathol.2007，31（1）:33-43.
[21］Benevolo M，Vocaturo A，Mottolese M，et al. Clinical role of p16INK4a expressionin liquid-based cervical cytology: correlation with HPV testing and histologicdiagnosis [J]. Am J Clin Pathol.2008,129(4):606-612.
[22］Mulvany NJ，Allen DG，Wilson SM. Diagnostic utility of p16INK4a: a reappraisalof its use in cervical biopsies[J］. Pathology.2008，40（4）:335-344.
1. Hannon GJ. RNA interference[J]. Nature.2002,418(6894):244-251.
2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics[J].CA Cancer J Clin.2011,61(2):69-90.
3. Madrigal M, Janicek MF, Sevin B, et al. In vitro antigene therapy targeting HPV-16E6and E7in cervical carcinoma [J].Gynecologic Oncology.1997,64（1）:18-25.
4. Reinhart BJ, Bartel DP. Small RNAs correspond to centromere heterochromaticrepeats[J]. Science.2002,297(5588),1831.
5. Grant SR. Dissecting the mechanisms of posttranscriptional gene silencing: Divide andconquer[J]. Cell.1999,96(3):303-306.
6. Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specificMicro-RNAs[J].Development cell.2003,5(2):351-358.
7. Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzymecomplex[J]. Science.2002,297(5589),2056-2060.
8. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X. R2D2, a bridgebetween the initiation and effector steps of the Drosophila RNAi pathway[J].Science.2003,301(5641):1921-1925.
9. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nucleasemediates post-transcriptional gene silencing in Drosophila cells[J]. Nature.2002,404(6775):293-296.
10. Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ, et al. Purified Argonaute2and an siRNA form recombinant human RISC[J]. Nat Struct Mol Biol.2005,12(4):340–9.
11. Plasterk RH. RNA silencing: the genome’s immune system[J]. Science.2002,296(5571):1263-1265.
12. Wei Q, Lipardi C, Paterson BM. Analysis of3’-hydroxyl group in Drosophila siRNAfunction [J].Methods.2003,30(4):337-347.
13. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference bydouble-standed RNA in Caenorhabditis elegans [J]. Nature.1998,391(6669):806-811.
14. Zhao Z, Cao Y, Li M, Meng A. Double-stranded RNA injection produces nonspecificdefects in zebrafish. Developmental Biology.2001,229(1):215-223.
15. Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCRproducts [J].RNA.2002,8(11):1454-1460.
16. Yu JY, Deruiter SL, Turner DL. RNA interference by expression of short-interferingRNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci USA.2002,99(9):6047-6052.
17. Sui G, Soohoo C,Affarel B, et al. A DNA vector-based RNAi technology to suppressgene expression in mammalian cells[J]. Proc Natl Acad Sci USA.2002,99(8):5515-5520.
18. Qin XF, An DS, Chen IS, et al. Inhabiting HIV-1infection in human T cells bylentiviral-medisted delivrey of small interfering RNA against CCR5[J]. Proc NatlAcad Sci USA.2003,100(1):183-188.
19. Liu CM, Lin DP, Dong WJ, et al. Retrovirus vector-mediated stable gene silencing inhuman cell [J]. Biochem Biophys Res Commun.2004,313(3):716-720.
20. Miyagishi M, Taira K.U6promoter-driven siRNAs with four uridine3’ overhangseffciently suppress targeted gene expression in mammmalian cells [J]. NatureBiotrchnology.2002,20(5):497-500.
21. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells [J]. Science.2002,296(5567):550-553.
22. Shen C, Buck AK, Liu X, et al.Gene silencing by adenovirus-delivered siRNA [J].FEBS Lett.2003,539(1-3):111-114.
23. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable genesilencing by RNAi in primary cells [J]. RNA.2003,9(4):493-501.
24. Tiscormia G, Singer O, Ikawa M, et al.A general method for gene knockdown in miceby using lentiviral vectors expression small interfering RNA [J]. Proc Natl Acad SciUSA.2003,100(4):1844-1848.
25. Elbashir SM, Harborth J,Lendeckel W, et al. Duplexes of21-nucleotide RNAs mediateRNA interference in mammalian cell culture [J]. Nature.2001,411(6836):494-498.
26. Harbotrh J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structuralvariation of small interfering RNAs and short hairpin RNAs and the effect onmammalian gene silencing [J]. Antisense Nucleic Acid Drug Dev.2003,13(2):83-105.
27. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J].Nat Biotechnol.2004,22(3):326-330.
28. Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targatedagainst HIV-1rev transcripts in human cells. Nature biotechology.2002,20(5):500-5.
29. Chen SH, Zhao G. Potential clinical applications of siRNA technique: benefits andlimitations. European journal of Clinical investigation[J]. Eur J Clin Invest.2011,41(2):221-32.
30. Zanier K, Charbonnier S, Baltzinger M, et al. Kinetic analysis of the interactions ofhuman papillomavirus E6oncoproteins with the ubiquitin ligase E6AP using surfaceplasmon resonance[J].J Mol Biol.2005,349(2):401-412.
31. Massimi P, Shai A, Lambert P, et al. HPV E6degradation of p53and PDZcontaining substrates in an E6AP null background[J].Oncogene.2008,27(12):1800-1804.
32. Sherman L, Alloul N, Golan I, et al.Expression and splicing patterns of humanpapillomavirus type-16mRNAs in pre-cancerous lesions and carcinomas of the cervix,in human keratinocytes immortalized by HPV16, and in cell lines established fromcervical cancers[J]. Int J Cancer.1992,50(3):356-64.
33. Sima N, Wang W, Kong D, et al. RNA interference against HPV16E7oncogene leadsto viral E6and E7suppression in cervical cancer cells and apoptosis via upregulationof Rb and p53[J]. Apoptosis.2008,13(2):273-81.
34. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive humancervical carcinoma cells treated with siRNA, a primer of RNA interference[J].Oncogene.2002,21(39):6041-8.
[2] Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer[J]. Clin Sci (Lond),2006,110(5):525-541.
[3] Tavassoli FA, Davilee P. WHO classification of tumor. Pathology&genetics tumors ofthe breast and female genital organs[M] Lion: ARC Press,2003:266.
[4] Cox JT. Human papillomavirus testing in primary cervical screening and abnormalPapanicolaou management [J]. Obstet Gynecol Surv,2006,61(6): S15-25.
[5] Wei Wang, Ni Sima, Debo Kong,et al. Selective targeting of HPV-16E6/E7in cervicalcancer cells with a potent oncolytic adenovirus and its enhanced effect withradiotherapy in vitro and vivo[J].Cancer Letters,2010,291(1):67-75.
[6] Hong D, Lu W, Ye F, et al. Gene silencing of HPV16E6/E7induced bypromoter-targeting siRNA in SiHa cells, Br J Cancer,2009,101(10):1798-1804.
[7] Murphy N, Ring M, Killalea AG, et al. p16INK4A as a marker for cervicaldyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears [J]. J ClinPathol,2003,56(1):56-63.
[8] Klaes R, Benner A, Friedrich T, et al. p16INK4a immunchistochemis-try inprovesinterobserver agement in the diagnosis of cervical inteaepthelial neoplasis [J]. Am JSurg Pathol,2002,26(11):1389-1399.
[9] Mutirangura A, Sriuranpong V, Termrunggraunglert W, et al. Telomerase activity andhuman papillomavirus in malignant, premalignant and benign cervical lesions [J]. Br JCancer,1998,78(7):933-939.
[10] Kailash U, Soundararajan CC, Lakshmy R, et al. Telomerase activity as an adjunct tohigh-risk human papillomavirus types16and18and cytology screening in cervicalcancer. Br J Cancer,2006,95(9):1250-7.
[11] Tu Z, Zhang AP, Wu RF, et al. Genomic amplification of the human telomeraseRNA gene for differential diadnosis of cervical disorder [J]. Cancer Genet Cytogen,2009,191(1):10-16.
[12] Gonzalez VM, Fuertes MA, Alonso C, et al. Is cisplatin-induced cell death alwaysproduced by apoptosis?[J]Mol Pharmacol,2001,59(4):657-663.
[13] Losa JH, Parada Cobo C, Viniegra JG, et al.Role of the p38MAPK pathway incisplatinbased therapy [J]. Oncogene2003,22(26):3998-4006.
[1] Tuschl T, Borkhardt A. Small interfering RNAs:a revolutionary tool for the analysis ofgene function and gene therapy[J]. Mol Interv,2002;2(3):158-67.
[2] Boutla A, Delidakis C, Livadaras I, et al. Variations of the3’protruding ends insynthetic short interfering RNA(siRNA) tested by microinjection in Drosophilaembryos [J]. Oligonucleotides,2003;13(5):295-301.
[3] Balsitis S, Dick F, Lee D, et al. Examination of the pRb-dependent andpRb-independent functions of E7in vivo.J Virol,2005,79(17):11392-402.
[4] Shai A, Brake T, Somoza C, Lambert PF. The human papillomavirus E6oncogenedysregulates the cell cycle and contributes to cervical carcinogenesis through twoindependent activities. Cancer Res,2007,67(4):1626-35.
[5] Elbashir SM, Harborth J, Lendckel W, et al. Duplexes of21-nucleotide RNAs mediateRNA interference in cultured mammalian cells [J]. Nature,2001;4119(6836):494-498.
[6] Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs formediating efficient RNAi in Drosophila melanogaster embryolysate [J]. EMBO J,2001,20(23):6877-88.
[7] Peter MW, Ming-Bo W, Tony L. Gene silencing as an adaptive defence against viruses[J]. Nature,2001,411(14):8342842.
[8] van Rij RP, Andino R. The silent treatment: RNAi as a defense against virus infectionin mammals [J]. Trends Biotechnol,2006,24(4):186-93.
[9] Schütz S, Sarnow P. Interaction of viruses with the mammalian RNA interferencepathway [J]. Virology,2006,5;344(1):151-7.
[10] Masiero M,Nardo G, Indraccolo S. RNA interference: implications for cancertreatment [J]. Mol Aspects Med,2007,28(1):143-166.
[11] Fewell GD, Schmitt K. Vector-based RNAi approaches for stable, inducible andgenome-wide screens [J]. Drug Discov Today,2006,11(21-22):975-82.
[12] Jiang M, Milner J. Selective Silencing of Viral Gene E6and E7Expression inHPV-Positive Human Cervical Carcinoma Cells Using Small Interfering RNAs [J].Methods Mol Biol,2004,292:401-20.
[13] Cho WC, Haryoung P, Young SC, et al. HPV E6antisense induces apoptosis in CaSkicells via suppression of E6splicing [J]. Exp Mol Med,2002,34(2):159-166.
[14] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells [J]. Science,2002,19;296(5567):550-3.
[15] Meissner JD. Nucleotide sequences and further characterization of humanpapillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma celllines [J]. Gen Virol,1999,80(pt7):1725-33.
[1] DiPaolo JA, Alvarez-Salas LM. Advances in the development of therapeutic nucleicacids against cervical cancer [J]. Expert Opin Biol Ther,2004,4(8):1251-57.
[2] Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application[J].Nat Rev Cancer,2002,2(5):342-50.
[3] Butz K, Ristriani T, Hengstermann A, et al. siRNA targeting of the viral E6oncogeneefficiently kills human papillomavirus-positive cancer cells [J]. Oncogene,2003,22(38):5938-45.
[4] Madrigal M, Janicek MF,Sevin B,et al.Averette HE.In vitro antigene therapy targetingHPV-16E6and E7in cervical carcinoma.Gynecologic [J]. Oncology.1997,61:18-25.
[5] Jiang M, Milner J. Selective Silencing of Viral Gene E6and E7Expression inHPV-Positive Human Cervical Carcinoma Cells Using Small Interfering RNAs [J].Methods Mol Biol.2004,292:401-20.
[6]吴剑，藤国英.中药诱导肿瘤细胞凋亡的研究进展［J］.国际医药卫生导报，2007,13(8):114-117．
[7] Lee KH, Yim EK, Kim CJ, et al. Proteomic analysis of anticancer effects by paclitaxeltreatment in cervical cancer cells[J]. Gynecol Oncol，2005，98(1):45-53．
[8] Forsmo S，Buhaug H，Skjeldestad FE，et al. Treatment of pre-invasive conditionsduring opportunistic screening and its effectiveness on cervical cancer incidence inone Norwegian county[J]. Int J Cancer,1997,71(1):4-8．
[9]豆长明,李继承.天花粉蛋白对HepA-H细胞和Hela细胞抑癌活性研究[J].中国病理生理杂志,2005,21(5):980-984．
[10]齐玉环,吴丹.细胞凋亡和p53在宫颈癌及宫颈上皮内瘤变中的表达[J].现代妇产科学进展,2009,9(1):16-18．
[11] Wang X, Martindale JL, Holbrook NJ. Requirement for ERK activation incisplatin-induced apoptosis [J]. J Biol Chem,2000,275(50):39435-39443.
[12]廖英俊，汤浩，金亚平.抗癌药顺铂对小鼠的耳、肾和肝毒性及其机制的研究[J].中国药理学通报,2004,(1):82-85.
[13]丁大连.顺铂及其耳毒性[J].中华耳科学杂志,2008,6(2）:125-133.
[14]柳萌,杨迪生.顺铂耐药机制的研究进展[J].现代中西医结合杂志，2007，16（6）:856-858.
[15]陈道桢,耿金花,薛文群,等.染色体不稳定性及其与妇科肿瘤形成关系的研究进展[J].东南大学学报（医学版），2004,23:355-357．
[16] Umayahara K, Numa F, Suehiro Y, et al. Comparative genomic hybridization detectsgenetic alteration during early stages of cervical cancer progression [J]. GenesChromosomes Cancer,2002,33:98-102.
[17] Kloth JN, Osting J, van Wezel T, et al. Combined arraycomparative genomichybridization and single-nucleotidepolymorphism-loss of heterozygosity analysisreveals complex genetic alterations in cervical cancer[J]. BMC Genomic,2007,8(20):53-66.
[18] Kerstin HH, Viktor J, Philip E, et al. Detection of genomic amplification of the humantelomerase gene (hTERC) in cytologic specimens as a genetic test for the diagnosis ofcervical dysplasia[J]. American Journal of Pathology,2003,163(4):1406-1416.
[1] Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential astherapeutics [J]. Nat Rev Drug Discov,2004,3(4):318-329.
[2] Lundstrom K. Prospects of treating neurological diseases by gene therapy[J]. Curr OpinInvestig Drugs,2007,8(1):34-40.
[3]廖维甲,梅铭惠,徐静,等.小干扰RNA抑制肝癌细胞株BEL7402ICAM-1基因表达的研究[J].中国现代医学杂志,2006,16(20):3065-3068.
[4] Yamada Y, Hamajima N, Kato T, et al. Association of a polymorphism of thephospholipase D2gene with the prevalence of colorectal cancer[J].J Mol Med,2003;81(2):126-131.
[5] Chen JQ, Zhan WH, He YL, et al. Expression of heparanase gene, CD44v6, MMP-7and nm23protein and their relationship with the invasion and metastasis of gastriccarcinomas[J].World J Gastroenterol,2004;10(6):776-782.
[6] Mcmanus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J].Nat.Rev Genet,2002;3(10):737-747.
[1] Andraw issm. Cervical cancer vaccines available in2007[J].Drug Discov Today,2005,10(14):949-950.
[2] Jemal A, Siegel R, Ward E, et al. Cancer statistics,2008[J]. CA Cancer J Clin,2008,58(2):71-96.
[3] Bulk S,Berkhof J,Bulkmans NW,et al.Preferential risk of HPV16for squamous cellcarcinoma and of HPV18for adenocarcinma of the cervix compared to woman withnormal normal cytology in the Netherlans[J] Br J Cancer,2006,94(1):171-175.
[4] Clifford GM, Gallus S, Herrero R, et al. Worldwide distribution of humanpapillomavirus types in cytologically normal woman in the International Agency forResearch on Cancer HPV prevalence surveys: a pooled analysis[J]. Lancet,2005;366(9490):991-998.
[5] Woodman CB, Collins S, Winter H, et al. Natural history of cervical humanpapillomavirus infection in young women: a longitudinal cohorts study[J]. Lancet,2001,357(9271):1831-1836.
[6] Plummer M, Schiffman M, Castle PE, et al. A2-year prospective study of humanpapillomavirus persistence among women with a cytological diagnosis of atypicalsquamous cells of undetermined significance or low-grade squamous intraepitheciallesion[J]. J Infect Dis,2007,195(11):1560-1562.
[7] Bulkmans NW, Berkhof J, Bulk S, et al. High-risk HPV type-specific clearance rates inCervical screening[J]. Br J Cancer,2007,96(9):1419-1424.
[8] Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer[J]. Clin Sci (Lond),2006,110(5):525-541.
[9] Tavassoli FA, Davilee P. WHO classification of tumor. Pathology&genetics tumors ofthe breast and female genital organs [M]. Lion: ARC Press,2003,266.
[10] Cox JT. Human papillomavirus testing in primary cervical screening and abnormalpapanicolaou management[J]. Obstet Gynecol Surv,2006,61(6):15-25.
[11] Siivia DS, Mireia D, Xavier C, et al. Worldwide prevalence and genotype distributionof cervical human papillomavirus DNA in women with normal cytology: ameta-analysis[J]. Lancet Infect Dis,2007,7(7):453-459.
[12] Castellsague X, Diaz M, Sanjose S, et al. Worldwide human papillomavirus etiologyof cervical adenocarcinoma and its cofactors: implication for screening andprevention [J]. Natl Cancer Inst,2006,98(5):303-315.
[13] Park JS, Ryu HS, Chang SJ, et al. The association of the cervical intraepithelialneoplasia and Human papillomavirus viral Load[J]. Korean J Obstet Gynecol,2005；48（28）：88-95.
[14] Yong MK，Jim YP，Kyung ML，et al. Does pretreatment HPV viral load correlate withprognosis in patients with early stage cervical carcinoma [J]. Gynecol Oncol,2008,19(2):113-116.
[15] Cheung JL, Cheung TH, Ng CW, et al. Analysis of human papillomavirus type18load and integration status from low-grade cervical lesion to invasive cervical cancer[J]. Journal of Clinical Microbiology,2009;47(2):287-293.
[16] Dalstein V, Riethmuller D, Prétet JL, et al. Persistence and load of high-risk HPV arepredictors for development of high-grade cervical lesions: A longitudinal Frenchcohort study[J]. Int J Cancer,2003,106(3):396-403.
[17] Akhtar M，Tu JJ.Human papilloma virus (HPV) vaccines：a major advance againstcervical cancer[J]. Adv Anat Pathol,2006,13(6):336-337.
[18] Villa LL, Costa RL, Petta CA, et al. Prophylactic quadrivalent human papillomavirus(types6,11,16,and18) L1virus-like particle vaccine in young women: a randomiseddouble-blind placebo-controlled multi-centre phase Ⅱ efficacy trial[J]. Lancet Oncol,2005,6(5):271-278.
[19] Harper DM, Franco EL, Wheeler CM, et al. Sustained efficacy up to4.5years of abivalent L1virus-like particle vaccine against human papillomavirus types16and18:follow-up from a randomized control trial[J]. Lancet,2006,367(9518):1247-1255.
[1] Garnett GP, Kim JJ，French K，et a1．Chapter21modelling the impactof HPVvaccinesion cervical cancer and screening programmes [J]. Vaccine.2006，24[Suppl3]：178-186．
[2] Cole ST, Danos O. Nucleotide sequence and comparative analysis of the humanpapillomavirus type18genome. Phylogeny of papillomaviruses and repeatedstructure of the E6and E7gene products [J]. J Mol Biol.1987,193(4),599–608.
[3] Keegan H, Mc Inerney J, Pilkington L, et al. Comparison of HPV detectiontechnologies: Hybrid capture2, PreTect HPV-Proofer and analysis of HPV DNAviral load in HPV16, HPV18and HPV33E6/E7mRNA positive specimens[J]. JVirol Methods.2009,155(1):61-66.
[4] Molden T, Kraus I, Skomedal H, et al. PreTect HPV-Proofer: real-time detection andtyping of E6/E7mRNA from carcinogenic human papillomaviruses[J]. J VirolMethods.2007,142(1-2):204-212.
[5] Zanier K, Charbonnier S, Baltzinger M, et al. Kinetic analysis of the interactions ofhuman papillomavirus E6oncoproteins with the ubiquitin ligase E6AP usingsurface plasmon resonance[J].J Mol Biol,2005,349(2):401-412.
[6] Massimi P, Shai A, Lambert P, et al. HPV E6degradation of p53and PDZcontaining substrates in an E6AP null background [J].Oncogene,2008,27(12):1800-1804.
[7] Camus S, Higgins M, Lane DP, et al. Differences in the ubiquitination of p53byMdm2and the HPV protein E6[J]. FEBS Lett.2003;536(1-3):220-224.
[8] Türkcüoglu I， Tezcan S，Kaygusuz G，et al. The role of p53，Bcl-2and Ki-67inpremalignant cervical lesions and cervical cancer［J. Eur J Gynaecol Oncol，2007，28（4）:290-293.
[9] Looi ML，Dali AZ，Ali SA，et al. Expression of p53，bcl-2and Ki-67in cervicalintraepithelial neoplasia and invasive squamous cellcarcinoma of the uterine cervix［J］. Anal Quant Cytol Histol.2008,30（2）:63-70.
[10] Liang CW, Lin MC, Hsiao CH，et al. Papillary squamous intraepithelial lesions ofthe uterine cervix: human papillomavirus-dependent changes in cell cycleexpression and cytologic features[J]. Hum Pathol.2010,41(3):326-335.
[11] Kruse AJ，Skaland I，Munk AC，et al. Low p53and retinoblastoma proteinexpression in cervical intraepithelial neoplasia grade3lesions is associated withcoexistent adenocarcinoma in situ［J］. Hum Pathol.2008，39（4）:573-578.
[12] Liu X， Clements A, Zhao K， etal. Structure of the human Papillomavirus E7oncoprotein and its mechanism for inactivation of the retinoblastoma tumorsuppressor[J]. J Biol Chem.2006,281(1)：578–586.
[13] Fiedler M, Müller-Holzner E, Viertler HP，et al. High level HPV-16E7oncoproteinexpression correlates with reduced pRb-levels in cervical biopsies.[J]. FASEB J.2004,18(10):1120-1122.
[14] Balsitis S, Dick F, Dyson N, Lambert PF. Critical roles for non-pRb targets ofhuman papillomavirus type16E7in cervical carcinogenesis[J]. Cancer Res.2006,66(19):9393-9400.
[15] Ishikawa M, Fujii T, Saito M, et al. Overexpression of p16IN K4A as an indicatorfor human papillomavirus oncogenic activity in cervical squamous neoplasia [J].Int J Gynecol Cancer.2006,16(l):347-353.
[16] Kalof AN, Evans MF, Simmons AL, et al. p16IN K4A immuno-expression andHPV in situ hybridization signal patterns: Potential markers of high-grade cervicalintraepithelial neoplasia [J]. Am J Surg Pathol.2005,29(5):674-679.
[17] Kanao H, Enomoto T, Ueda Y, et a l. Correlation betw een pl4/p16IN K4Aexpression and HPV infection in uterine cervical cancer [J]. Cancer Lett.2004,213(1):31-37.
[18］Longatto FA，Etlinger D，Pereira SM，et al. The association of p16INK4A andfragile histidine triad gene expression and cervical lesions[J]. J Low Genit Tract Dis.2007，11（3）:151-157.
[19］Redman R, Rufforny I，Liu C，et al. The utility of p16（Ink4a） in discriminatingbetween cervical intraepithelial neoplasia1and nonneoplastic equivocal lesions ofthe cervix [J]. Arch Pathol Lab Med.2008,132（5）:795-799.
[20］Kong CS， Balzer BL， Troxell ML, et al. p16INK4A immunohistochemistry issuperior to HPV in situ hybridization for the detection of high-risk HPV in atypicalsquamous metaplasia [J]. Am J Surg Pathol.2007，31（1）:33-43.
[21］Benevolo M，Vocaturo A，Mottolese M，et al. Clinical role of p16INK4a expressionin liquid-based cervical cytology: correlation with HPV testing and histologicdiagnosis [J]. Am J Clin Pathol.2008,129(4):606-612.
[22］Mulvany NJ，Allen DG，Wilson SM. Diagnostic utility of p16INK4a: a reappraisalof its use in cervical biopsies[J］. Pathology.2008，40（4）:335-344.
1. Hannon GJ. RNA interference[J]. Nature.2002,418(6894):244-251.
2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics[J].CA Cancer J Clin.2011,61(2):69-90.
3. Madrigal M, Janicek MF, Sevin B, et al. In vitro antigene therapy targeting HPV-16E6and E7in cervical carcinoma [J].Gynecologic Oncology.1997,64（1）:18-25.
4. Reinhart BJ, Bartel DP. Small RNAs correspond to centromere heterochromaticrepeats[J]. Science.2002,297(5588),1831.
5. Grant SR. Dissecting the mechanisms of posttranscriptional gene silencing: Divide andconquer[J]. Cell.1999,96(3):303-306.
6. Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specificMicro-RNAs[J].Development cell.2003,5(2):351-358.
7. Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzymecomplex[J]. Science.2002,297(5589),2056-2060.
8. Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X. R2D2, a bridgebetween the initiation and effector steps of the Drosophila RNAi pathway[J].Science.2003,301(5641):1921-1925.
9. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nucleasemediates post-transcriptional gene silencing in Drosophila cells[J]. Nature.2002,404(6775):293-296.
10. Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ, et al. Purified Argonaute2and an siRNA form recombinant human RISC[J]. Nat Struct Mol Biol.2005,12(4):340–9.
11. Plasterk RH. RNA silencing: the genome’s immune system[J]. Science.2002,296(5571):1263-1265.
12. Wei Q, Lipardi C, Paterson BM. Analysis of3’-hydroxyl group in Drosophila siRNAfunction [J].Methods.2003,30(4):337-347.
13. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference bydouble-standed RNA in Caenorhabditis elegans [J]. Nature.1998,391(6669):806-811.
14. Zhao Z, Cao Y, Li M, Meng A. Double-stranded RNA injection produces nonspecificdefects in zebrafish. Developmental Biology.2001,229(1):215-223.
15. Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCRproducts [J].RNA.2002,8(11):1454-1460.
16. Yu JY, Deruiter SL, Turner DL. RNA interference by expression of short-interferingRNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci USA.2002,99(9):6047-6052.
17. Sui G, Soohoo C,Affarel B, et al. A DNA vector-based RNAi technology to suppressgene expression in mammalian cells[J]. Proc Natl Acad Sci USA.2002,99(8):5515-5520.
18. Qin XF, An DS, Chen IS, et al. Inhabiting HIV-1infection in human T cells bylentiviral-medisted delivrey of small interfering RNA against CCR5[J]. Proc NatlAcad Sci USA.2003,100(1):183-188.
19. Liu CM, Lin DP, Dong WJ, et al. Retrovirus vector-mediated stable gene silencing inhuman cell [J]. Biochem Biophys Res Commun.2004,313(3):716-720.
20. Miyagishi M, Taira K.U6promoter-driven siRNAs with four uridine3’ overhangseffciently suppress targeted gene expression in mammmalian cells [J]. NatureBiotrchnology.2002,20(5):497-500.
21. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of shortinterfering RNAs in mammalian cells [J]. Science.2002,296(5567):550-553.
22. Shen C, Buck AK, Liu X, et al.Gene silencing by adenovirus-delivered siRNA [J].FEBS Lett.2003,539(1-3):111-114.
23. Stewart SA, Dykxhoorn DM, Palliser D, et al. Lentivirus-delivered stable genesilencing by RNAi in primary cells [J]. RNA.2003,9(4):493-501.
24. Tiscormia G, Singer O, Ikawa M, et al.A general method for gene knockdown in miceby using lentiviral vectors expression small interfering RNA [J]. Proc Natl Acad SciUSA.2003,100(4):1844-1848.
25. Elbashir SM, Harborth J,Lendeckel W, et al. Duplexes of21-nucleotide RNAs mediateRNA interference in mammalian cell culture [J]. Nature.2001,411(6836):494-498.
26. Harbotrh J, Elbashir SM, Vandenburgh K, et al. Sequence, chemical, and structuralvariation of small interfering RNAs and short hairpin RNAs and the effect onmammalian gene silencing [J]. Antisense Nucleic Acid Drug Dev.2003,13(2):83-105.
27. Reynolds A, Leake D, Boese Q, et al. Rational siRNA design for RNA interference [J].Nat Biotechnol.2004,22(3):326-330.
28. Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targatedagainst HIV-1rev transcripts in human cells. Nature biotechology.2002,20(5):500-5.
29. Chen SH, Zhao G. Potential clinical applications of siRNA technique: benefits andlimitations. European journal of Clinical investigation[J]. Eur J Clin Invest.2011,41(2):221-32.
30. Zanier K, Charbonnier S, Baltzinger M, et al. Kinetic analysis of the interactions ofhuman papillomavirus E6oncoproteins with the ubiquitin ligase E6AP using surfaceplasmon resonance[J].J Mol Biol.2005,349(2):401-412.
31. Massimi P, Shai A, Lambert P, et al. HPV E6degradation of p53and PDZcontaining substrates in an E6AP null background[J].Oncogene.2008,27(12):1800-1804.
32. Sherman L, Alloul N, Golan I, et al.Expression and splicing patterns of humanpapillomavirus type-16mRNAs in pre-cancerous lesions and carcinomas of the cervix,in human keratinocytes immortalized by HPV16, and in cell lines established fromcervical cancers[J]. Int J Cancer.1992,50(3):356-64.
33. Sima N, Wang W, Kong D, et al. RNA interference against HPV16E7oncogene leadsto viral E6and E7suppression in cervical cancer cells and apoptosis via upregulationof Rb and p53[J]. Apoptosis.2008,13(2):273-81.
34. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive humancervical carcinoma cells treated with siRNA, a primer of RNA interference[J].Oncogene.2002,21(39):6041-8.