靶向survivin的小干扰RNA对HCCLM6细胞恶性表型的影响  

Effect of siRNA targeted against survivin on the malignant behaviors of human hepatoma cells

作　　者：韩丹[1] 叶胜龙[1] 刘彬彬[1] 陈荣新[1] 薛同春[1] 孙瑞霞[1] 赵燕[1] 陈洁[1] 

机构地区：[1]复旦大学肝癌研究所、复旦大学中山医院、教育部癌变与侵袭原理重点实验室,上海200032

出　　处：《中华肝脏病杂志》2008年第5期363-366,共4页Chinese Journal of Hepatology

基　　金：国家重点基础研究发展规划（973）项目（2004CB518708）

摘　　要：目的研究survivin基因在肝癌细胞中的表达水平及survivin小干扰RNA对高侵袭潜能人肝癌细胞HCCLM6恶性表型的影响。方法收集4种人肝癌细胞株，以RT—PCR和Westernblot检测survivin蛋白质表达，进而筛选出高表达survivin基因的肝癌细胞。构建针对survivinmRNA的干扰质粒pshRNAsurvivin及阴性对照质粒pGPU6／GFP／Neo-NC，并将其转染HCCLM6细胞。实时荧光定量PCR检测survivinmRNA表达量的变化情况；肿瘤细胞黏附实验检测细胞黏附能力的改变及Matrigel侵袭实验检测细胞侵袭力的改变。结果4种肝癌细胞随侵袭能力升高survivinmRNA表达水平依次升高（P〈0．05），HCCLM6细胞survivin表达水平最高；Westernblot结果与RT—PCR结果一致。HCCLM6细胞转染pshRNAsurvivin后，survivinmRNA表达量明显下降，抑制率达93．500％±3．117％，与转染阴性对照质粒组（8．215％±0．797％）相比，差异有统计学意义（t=70．852，P〈0．01）。细胞黏附实验结果显示，survivin重组质粒转染组黏附率为11．403％±1．256％，阴性对照组为32．545％±1．367％（t=20．732，P〈0．01）。Matrigel侵袭实验结果显示，与阴性对照组【（32．6±1．4）】个相比，转染pshRNA survivin后，细胞侵袭力明显下降【（13．5±0．9）个，t=14．5，P〈0．01】。结论Survivin与肝癌侵袭转移等恶性生物学行为有关，并随侵袭转移潜能升高表达水平升高。pshRNAsurvivin可以特异性抑制高侵袭肝癌细胞HCCLM6中survivin表达，并显著降低其侵袭、黏附能力，小干扰RNA技术有望成为抑制肝癌侵袭转移的新途径。Objectives To study survivin expression in human hepatoma cells and the effects of survivin siRNA on the malignant phenotypes of human hepatocellular cell line HCCLM6. Methods Four hepatocellular carcinoma （HCC） cell lines were used. Semi-quantitative RT-PCR and Western blot were used to measure and compare their survivin expressions. The siRNA expression vector pshRNA-survivin targeting the mRNA of survivin and vector pGPU6/GFP/Neo-NC （as a control） were constructed, and then transfected into HCCLM6 cells, FQ-PCR was used to quantify the mRNA levels of survivin. The malignant phenotypes of transfected HCCLM6 cells, including invasive activities and adhesive capabilities, were analyzed. Results Survivin expression gradually increased with the increase of the invasion and metastasis behaviors of the four HCC cell lines （P 〈 0.05）. The expression of survivin was highest in cell line HCCLM6. Survivin mRNA level was decreased by 93.500% ± 3.117% after the pshRNA-survivin transfection, The cell adhesion rates significantly decreased in the cells transfected with pshRNA-survivin （cell adhesion rates were 11.403% ± 1.256% vs 32.545% ± 1.367%, t = 20.732, P 〈 0.01）. The migrating number of HCCLM6 cells （13.5 ± 0.9）transfected with pshRNA-survivin was also significantly decreased （t = 14.5, P 〈 0.01） as compared with the control group （32.6 ± 1.4）. Conclusion The expression of survivin in HCC might have a close relationship to their invasion and metastasis properties. Sequence-specific shRNA can significantly reduce the survivin expression in the HCCLM6 cell line. Suppression of survivin expression in HCCLM6 cells transfected with pshRNA-survivin can reduce their invasive and adhesive capabilities.

关 键 词：癌 肝细胞 RNA干扰 SURVIVIN 

分 类 号：R735.7[医药卫生—肿瘤]