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反转录病毒介导的HPV16 E6-siRNA对SiHa细胞增殖及抗药性的影响
         
Influence of retrovirus-mediated HPV16 E6-siRNA on proliferation and drug-resistance of SiHa cells

摘    要
目的: 探讨反转录病毒介导的靶向人乳头瘤病毒(human papillomavirus,HPV)16E6基因的小干扰RNA(small interfering RNA,siRNA)对HPV16阳性宫颈癌SiHa细胞的影响。方法: 收集产反转录病毒PA317细胞的病毒上清液感染SiHa细胞,G418筛选获得HPV16E6基因稳定沉默的细胞克隆(SiHa/16E6),同时设立载体对照(SiHa/pSUPER)和正常细胞对照(SiHa)。RT-PCR检测HPV16 E6和E7 mRNA的表达水平,Western印迹法检测p53、Rb、caspase 3及其底物多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)蛋白的表达,MTT法检测细胞的增殖活性及细胞对顺铂的敏感性。结果: 感染后与SiHa细胞对照组相比,各时间点的E6和E7 mRNA表达水平均有所下降,p53和Rb蛋白表达水平增高,caspase-3和PARP剪切后的活性条带增加,感染后40 d细胞的增殖活性下降;在同一药物浓度下,感染后20 d的细胞存活率显著降低,顺铂的IC50值为(3.92±2.48) μg/mL。而SiHa/pSUPER组与SiHa细胞对照组相比,差异无统计学意义。结论: 反转录病毒介导的HPV16 E6-siRNA可有效沉默SiHa细胞中E6和E7基因的表达,抑制细胞增殖,并提高细胞对顺铂的敏感性。
标    签 宫颈肿瘤   RNA干扰   反转录病毒科   人乳头状瘤病毒16   基因表达   细胞增殖   抗药性   Uterine cervical neoplasms   RNA interference   Retroviridae   Human papillomavirus 16   Gene expression   Cell proliferation   Drug resistance  
 
Abstract
Objective: To investigate the effects of retrovirus-mediated siRNA targeting human papillomavirus (HPV)16E6 gene (HPV16 E6-siRNA) on the HPV 16-positive cervical cancer SiHa cells. Methods: The SiHa cells were infected with the supernatant collected from the retrovirus-generating PA317 cells and then selected by G418 screening to obtain HPV16E6 gene-silencing cell clone (SiHa/16E6). The control groups (SiHa/pSUPER group and SiHa group) were set up concurrently. The mRNA levels of HPV16 E6 and E7 were tested by RT-PCR; the protein expressions of p53, Rb, caspase 3 and its substrate PARP were detected by Western blotting; MTT assay was performed to measure the proliferation activity of cells and their sensitivity to cisplatin. Results: Compared with SiHa group, E6 and E7 mRNA expressions were decreased, while p53 and Rb protein expressions were increased at each time point after transfection. The cleaved strips of both caspase-3 and PARP were increased and the proliferation activity of SiHa/16E6 cells was decreased on day 40 after infection. The survival rate of SiHa/16E6 cells were markedly decreased after 20-d infection and the IC50 value of cisplatin was (3.92±2.48)μg/mL, whereas the difference was not significant between SiHa/pSUPER group and SiHa group. Conclusion: The retrovirus-mediated HPV16 E6 siRNA effectively silenced the expression of E6 and E7 genes, inhibited cell proliferation, and increased the sensitivity of SiHa cells to cisplatin.

中图分类号 R737.33   DOI 10.3781/j.issn.1000-7431.2009.05.009

 
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所属栏目 基础研究

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收稿日期 2008/9/24

修改稿日期 2009/3/10

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备注葛翠翠(1983-),女(汉族),硕士研究生

引用该论文: GE Cui-cui,WANG Yan-kui,LUO Bing,WANG Wen-juan,YANG Ling. Influence of retrovirus-mediated HPV16 E6-siRNA on proliferation and drug-resistance of SiHa cells[J]. Tumor, 2009, 29(5): 438~442
葛翠翠,王言奎,罗兵,王文娟,杨玲. 反转录病毒介导的HPV16 E6-siRNA对SiHa细胞增殖及抗药性的影响[J]. 肿瘤, 2009, 29(5): 438~442


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