HIF-1α反义寡核苷酸对喉鳞状细胞癌hep-2细胞放疗的增效作用

Synergistic effect of HIF-1α antisense oligonucleotides on apoptosis and radiotherapeutic activity against hep-2 cells in vitro

目的:通过HIF-1α反义寡核苷酸转染hep-2细胞株,观察细胞凋亡及对放疗的反应情况。方法:体外培养人喉鳞状细胞癌hep-2细胞株,脂质体介导反义寡核苷酸转染,6 h后4 Gyγ射线照射。低氧培养24 h后RT-PCR检测HIF-1α mRNA表达,流式细胞仪检测HIF-1α蛋白水平及细胞凋亡率,MTT检测细胞存活情况。结果:低氧使人喉鳞状细胞癌hep-2细胞株对放射的敏感性下降;低氧并未影响HIF-1α mRNA的表达,HIF-1α蛋白在低氧6 h时表达已开始升高,低氧36 h时达高峰,之后表达逐渐下降;放射刺激对HIF-1α蛋白表达差异无统计学意义(P>0.05);各浓度的反义寡核苷酸转染组降低HIF-1α的转录及蛋白水平;MTT结果显示随反义核苷酸浓度增高,其增殖抑制效应越强(P<0.05),正义对照核苷酸及脂质体对照没有显著的抑制效应(P>0.05),但转染浓度在400 nmol/L以上时,正义对照也表现出一定的增殖抑制作用(P<0.05);放疗后反义寡核苷酸转染组蛋白表达下降明显,较对照组及单纯放射组细胞凋亡率增加(P<0.01)。结论:低氧环境下人喉鳞状细胞癌hep-2细胞株对放射有抵抗作用,这种抵抗作用与低氧时HIF-1α蛋白表达上调有关,而放射本身并未上调HIF-1α蛋白的表达;HIF-1α反义寡核苷酸转染hep-2细胞株能够降低HIF-1α蛋白表达,提高低氧环境下人喉鳞状细胞癌hep-2细胞株对放疗的敏感性。

Objective：To investigate the effect of HIF-1α down- regulation by antisense oligonucleotides （AS ODNs） on hep-2 cells apoptosis and radiotherapy sensitivity in vitro. Method： AS ODN was transfected into cultured hep-2 cells which received radiation under hypoxic condition. Twenty-four hours after hypoxic treatment cells were observed under the fluorescence inverse phase microscope. Apoptosis rate, expression of HIF-1αmRNA and protein were measured. Result： In cultured hep-2 cells, AS ODN down-regulated HIF-1αmRNA expression. The protein levels were lowered down and the cell viability were decreased. In contrast, the scrambled control ODN caused minimal HIF-1α loss and less cell inhibition. In addition, AS ODN sensitized Hep-2 cells to the radiation effects of 4Gy ^60Co ray. Conclusion： HIF-1α is a viable target for cancer therapy in human laryngeneal neoplasms. In cultured Hep-2 cells HIF-1α AS ODN can down-regulate HIF-1αprotein expression, induce apoptosis and enhance the radiotherapy sensitivity.