天津医药

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靶向抑制HIF-1α基因对舌癌细胞增殖活性的影响

徐香兰1,韩冰2,燕杰1,王丽1   

  1. 1. 天津医学高等专科学校病原生物与免疫学教研室
    2. 天津市第三中心医院耳鼻喉
  • 收稿日期:2012-11-26 修回日期:2013-05-05 出版日期:2013-09-15 发布日期:2013-09-15
  • 通讯作者: 徐香兰

Effect of RNA interference targeting HIF-1a gene on the proliferation of human tongue cancer cells

XU Xiang lan1, HAN Bing 2,YAN Jie 3,WANG Li 3   

  1. 1. Department of Pathogenic Biology and Immunology, Tianjin Medical Colleg
    2. Tianjin Third Central Hospital
    3. Department of Pathogenic Biology and Immunology, Tianjin Medical College
  • Received:2012-11-26 Revised:2013-05-05 Published:2013-09-15 Online:2013-09-15
  • Contact: XU Xiang lan

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摘要：

【摘要】 目的 研究靶向抑制乏氧诱导因子（HIF）-1α基因对Tca8113及Tscca舌癌细胞株增殖的影响。方法  将HIF-1α基因的短发夹干扰RNA（shRNA）的表达载体与空载质粒分别转染人舌癌细胞中作为siHIF-1α组和空载组，半定量RT-PCR及Western blot法检测HIF-1α的mRNA及蛋白表达。软琼脂克隆形成实验观察细胞的增殖；细胞计数法检测活细胞的数量；流式细胞分析术检测细胞周期的分布；同时将舌癌细胞接种至裸鼠皮下，观察体内的成瘤情况。结果 HIF-1αshRNA质粒转染舌癌细胞株后，HIF-1α的mRNA及蛋白表达明显下降。siHIF-1α组细胞在软琼脂中形成克隆的能力显著下降。细胞计数表明siHIF-1α组活细胞数量减少。转染后乏氧培养48h si?HIF-1α组细胞周期阻滞于G0/G1期。舌癌细胞移植到裸鼠体内数天后siHIF-1α组成瘤体积明显小于空载组（P＜0.05）。结论 HIF-1α基因能够促进舌癌细胞增殖活性，有可能成为未来治疗舌癌的一个潜在作用靶点。

关键词: 舌肿瘤, 细胞系, 肿瘤, RNA干扰, 缺氧诱导因子1, α亚基, 逆转录聚合酶链反应, 印迹法

Abstract:

[Abstract]   Objective  To explore the effect of RNA interferencetargeting hypoxia-inducible factor-1α(HIF-1α)
gene on the proliferation of human tongue cancer cells (Tca8113and Tscca).  Methods  HIF-1α- specific short hairpin
RNA (shRNA) expression vector and empty shRNA vector were respectively transferred into two kinds of cultured human tongue cancer cells. The expression of HIF-1αgene was detected by RT-PCR and Western blot method. The clone formation assay was used to observe the cell proliferation. The cell counting was used to monitor the number of living cells. Flow cytometry was applied to detect cell cycle distribution. Additionally, the cells were injected into nude mice, and tumor forming condition was observed.   Results   After the shRNA HIF-1αplasmid was transfected into tongue cancer cells, the expressions of both HIF-1αmRNA and protein were significantly decreased in tongue cancer cells under hypoxia condition. The number of colony formation was significantly lower in HIF-1αsmall interfering RNA (siRNA) group than that in empty and negative control group. The cell counting revealed that the number of living cells was remarkably lower in HIF-1αsiRNA groupthan that in negative control group. Also, the cell cycle in HIF-1αsiRNA group was arrested at G0/G1phase after transfection and 48hours hypoxia culture. Besides, the tumor volume wasremarkably smaller in HIF-1αsiRNA groupthan that in negative control group after thetongue cancer cells transplanted into nude mice for several days. The statistical analy sis indicated that the difference was significant (P＜0.05). Conclusion   RNA interference targeting HIF-1αgene could suppress the proliferation of human tongue cancer cells effectively. HIF-1αgene could be researched as a new therapeutic target of tongue cancer in the future.

Key words: tongue neoplasms, cell line, tumor, RNA interference, hypoxia-inducible factor 1, alpha subunit, reverse transcriptase polymerase chain reaction, 印迹法