Survivin downregulation by siRNA/cationic liposome complex radiosensitises human hepatoma cells in vitro and in vivo
Survivin downregulation by siRNA/cationic liposome complex radiosensitises human hepatoma cells in vitro and in vivo
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DOI：
10.3109/09553001003668006
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发表时间：
2010-05
影响因子：
2.6
通讯作者：
Wei Yang;T. Sun;Jianping Cao;Fenju Liu
中科院分区：
文献类型：
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作者：
Wei Yang;T. Sun;Jianping Cao;Fenju Liu
目的：探讨Survivin短发夹状RNA（shRNA）对人肝癌SMMC-7721细胞增殖、凋亡及放射敏感性的影响。材料与方法：构建Survivin靶向siRNA表达载体，经阳离子脂质体介导转染SMMC-7721细胞。采用逆转录-聚合酶链反应（RT-PCR）和Western blotting检测Survivin mRNA和蛋白表达。通过3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑（MTT）测定来测量细胞活力。流式细胞仪检测细胞周期和凋亡。采用集落形成试验检测SMMC-7721细胞的放射敏感性。皮下植入SMMC-7721细胞的小鼠在治疗后监测肿瘤生长和存活，并通过免疫组织化学染色分析肿瘤的增殖、凋亡和血管生成生物标志物。结果如下：转染后SMMC-7721细胞中Survivin基因mRNA和蛋白表达下调，细胞生长受到明显抑制，细胞阻滞于G2/M期，凋亡率增加，放射敏感性增加。Survivin-shRNA联合放疗在抑制肿瘤生长和延长生存期方面比单独放疗或Survivin-shRNA治疗更有效。联合治疗抑制细胞增殖和肿瘤血管生成，并增加肿瘤异种移植物的凋亡。结论：siRNA/阳离子脂质体下调Survivin表达抑制人肝癌细胞增殖、诱导凋亡和增强放射敏感性的实验研究
Purpose: To investigate the effect of survivin-short hairpin RNA (shRNA) on the proliferation, apoptosis and radiosensitivity of human hepatoma SMMC-7721 cells. Materials and methods: Survivin-targeted small interfering RNA (siRNA) expression vector was constructed and transfected into SMMC-7721 cells mediated by cationic liposome. Survivin mRNA and protein expression were analysed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell cycle and apoptosis were measured by flow cytometry (FCM) assay. Radiosensitivity of SMMC-7721 cells was examined using a colony-forming assay. Mice subcutaneously implanted with SMMC-7721 cells were monitored for tumour growth and survival after treatment, and tumours were analysed for proliferation, apoptosis, and angiogenesis biomarkers by immunohistochemistry staining. Results: After transfection, the mRNA and protein expression of survivin gene in SMMC-7721 cells downregulated, which led to significant cell growth inhibition, cell arrest in G2/M phase, increased apoptotic rate and radiosensitivity. Survivin-shRNA in combination with radiotherapy was more effective than radiotherapy or survivin-shRNA therapy alone in suppressing tumour growth and extending survival duration. Combined therapy inhibited cell proliferation and tumour angiogenesis and increased apoptosis in tumour xenografts. Conclusion: Survivin downregulation by siRNA/cationic liposome inhibited proliferation, induced apoptosis and enhanced radiosensitivity in human hepatoma cells in vitro and in vivo.