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特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

Inhibition of human papillomavirus type 16 E6 gene expression with HPV16 E6 specific siRNA in cervical carcinoma cell line
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摘要 目的优化HPV-16 E6癌基因特异的U6质粒表达的siRNA,抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列,合成DNA链,构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1.0-U6载体,导入HPV-16 DNA阳性的宫颈癌细胞株CaSki中,观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化,并观察细胞生长被抑制的情况。结果4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96 h内,可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低,其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。West- ern blot分析表明,4个HPV-16 E6 siRNA作用72 h后,未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢;选择针对E6内含子的siRNA作用位点,特异性抑制E6表达;而针对E6外显子的siRNA作用位点,可抑制E6和E7基因的表达,是用于治疗HPV阳性宫颈癌细胞的理想靶位。 Objective To study the HPV-16 E6 specific shRNA expressed by U6 plasmid to inhibit the expression of HPV oncogene and the growth of cervical cancer cell. Methods We selected 4 target mRNA sequences to the exon and interon of HPV-16 E6 mRNA, and synthesized dsDNA to construct the recombinant pSilencer1.0-U6 vector which expresses HPV-16 E6 short hairpin dsRNA. After transfected into HPV-16 DNA positive cervical cancer cell line CaSki, then observe the expression level of HPV-16 E6, E7 genes and their proteins as well as the inhibition effects of cell growth. Results The cellular growth velocity was down-regulated both by 4 HPV-16 E6 specific siRNA in 0-96 h after recombinant pSilencerl.0-U6 vector transfected into CaSki cell. The abundance of HPV-16 E6 E7 mRNA inside CaSki cells were also reduced by the4 HPV-16 E6 specific siRNA respectively, while 1 HPV-16 E6 specific siRNA targetfing to the interon sequence of E6 gene inhibited the HPV-16 E6 mRNA only. HPV-16 E6 protein was undetectable with Western blot assay 72 h later after 4 HPV-16 E6 specific siRNA transfected into CaSki cell. Conclusion It suggested that the growth of cervical cancer cells was inhibited by HPV-16 E6 specific siRNA. The HPV16 E6 specific siRNA targeting to the interon of E6 mRNA can inhibit the expression of HPV-16 E6 gene only, while the HPV-16 E6 specific siRNAs targeting to the exon of E6 mRNA can inhibit the expression of both E6 and E7 genes, which can be used as a good candidates of cancer therapeutical siRNA drug.
作者 栾怡 于修平 赵蔚明 周亚滨 白晓卉
机构地区 山东大学医学院微生物学教研室
出处 《中华微生物学和免疫学杂志》 CAS CSCD 北大核心 2006年第7期598-602,共5页 Chinese Journal of Microbiology and Immunology
基金 山东省科学技术发展计划重点项目(项目编号:2004GG2202105) 山东省卫生厅基金(项目编号:2003026)
关键词 人乳头状瘤病毒 siRNA 子宫颈肿瘤 细胞生长 Human papillomavirus siRNA Cervical carcinoma Cell growth
分类号 R737.33 [医药卫生—肿瘤]
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参考文献12

  • 1Leung RK,Whittaker PA.RNA interference:from gene silencing to gene-specific therapeutics.Pharmacol Ther,2005,107(2):222-239.
  • 2Rose BR,Thompson CH,Tattersall MH,et al.Identification of E6/E7transcription patterns in HPV 16-positive cervical cancers using the reverse transcription/polymerase chain reaction.Gynecol Oncol,1995,56(2):239-244.
  • 3Kang HT,Lee CJ,Seo EJ,et al.Transition to an irreversible stateofsenescence in HeLa cells arrested by repression of HPV E6 and E7 genes.Mech Ageing Dev,2004,125(1):31-40.
  • 4Sterlinko Grm H,Weber M.Inhibition of E6-induced degradation of its cellular substrates by novel blocking peptides.J Mol Biol,2004,335(4):971-985.
  • 5Yoshinouchi M,Yamada T,Kizaki M,et al.In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by E6 siRNA.Mol Ther,2003,8(5):762-768.
  • 6Braun K,Ehemann V,Waldeck W,et al.HPV18 E6 and E7 genes affect cell cycle,pRB and p53 of cervical tumor cells and represent prominent candidates for intervention by use peptide nucleic acids (PNAs).Cancer Lett,2004,209(1):37-49.
  • 7Lu DW,El-Mofty SK,Wang HL.Expression of p16,Rb and p53 proteins in squamous cell carcinomas of the anorectal region harboring human papillomavirus DNA.Mod Pathol,2003,16(7):692-699.
  • 8牛晓宇,彭芝兰,王和.RNA干涉抑制宫颈癌CaSki细胞株HPV16 E6基因的研究[J].癌症,2004,23(11):1257-1262. 被引量：27
  • 9钟绮丽,李建军,程滨珠,于洁.发夹状RNA对人乳头瘤病毒16型E6表达的影响[J].中华皮肤科杂志,2004,37(2):94-96. 被引量：1
  • 10Zheng ZM,Tao M,Yamanegi K,et al.Splicing of a cap-proximal human papillomavirus 16 E6E7 intron promotes E7 expression,but can be restrained by distance of the intron from its RNA 5' cap.J Mol Biol,2004,337(5):1091-1108.

二级参考文献26

  • 1Majewski S, Jablonska S. Human papillomavirus-associated tumors of the skin and mucosa. J Am Acad Dermatol, 1997, 36:659-685.
  • 2Majewski S, Jablonska S. Epidermodysplasia verruciformis as a model of human papillomavirus-induced genetic cancer of the skin.Arch Dermatol, 1995, 131:1312-1318.
  • 3Bosch FX, Manos MM, Munoz N, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst, 1995, 87:796-802.
  • 4Paul CP, Good PD, Winer I, et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol, 2002, 20:505-508.
  • 5Yin JQ, Wan Y. RNA-mediated gene regulation system: now and the future (Review). Int J Mol Med, 2002, 10:355-365.
  • 6Medina MF, Joshi S. RNA-polymerase Ⅲ-driven expression cassettes in human gene therapy. Curr Opin Mol Ther, 1999, 1:580-594.
  • 7Lee CJ,Suh EJ,Kang HT,et al.Induction of senescence like state and suppression of telomerase activity through inhibition of HPV E6/E7 gene expression in cells immortalized by HPV 16 DNA [J].Exp Cell Res,2002,277(2):173-182.
  • 8Tan TM,Ting RC.In vitro and in vivo inhibition of human papillomavirus type 16 E6 and E7 genes [J].Cancer Res,1995,55(20):4599-4605.
  • 9Choo CK,Ling MT,Suen CK,et al.Retrovirus mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of Caski cells [J].Gynecol Oncol,2000,78(3 pt 1):293-301.
  • 10Storey A,Oates D,Banks L,et al.Anti sense phosphorothioate oligonucleotides have both specific and non specific effects on cells containing human papillomavirus type 16 [J].Nucleic Acids Res,1999,19(15):4109-4114.

共引文献26

中华微生物学和免疫学杂志
2006年 第7期

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