小分子HIF-1α干扰RNA逆转鼻咽癌细胞耐药的实验

Experimental on Drug-resistant Effect on Nasopharyngeal Cancer Cells with siRNA-HIF-1α Silencing

目的探讨小分子HIF-1α干扰RNA对人鼻咽癌耐药细胞株CNE2/DDP耐药性的影响。方法采用药物大剂量冲击和逐渐增加剂量相结合的方法建立人鼻咽癌顺铂耐药细胞株CNE2/DDP,瞬时转染法将HIF-1α小片段RNA导入CNE2/DDP细胞沉默HIF-1α基因,MTS法及流式细胞仪分别检测顺铂对CNE2/DDP细胞增殖、凋亡的影响,Western blot法分析细胞HIF-1α及MDR1表达水平。结果成功建立了人鼻咽癌顺铂耐药细胞株CNE2/DDP,耐药系数达16。当顺铂≥1.0μg/ml时,CNE2组与siHIF-1αsiRNA组细胞抑制率均明显高于CNE2/DDP组与siCtrl-siRNA组(P<0.05);同样,CNE2组与siHIF-1αsiRNA组细胞凋亡率也明显高于CNE2/DDP组与siCtrl-siRNA组(P<0.05);Westernblot显示siHIF-1αsiRNA组HIF-1α及MDR1蛋白的表达低于CNE2/DDP组。结论小分子HIF-1α干扰RNA沉默HIF-1α提高了人鼻咽癌顺铂耐药细胞CNE2/DDP对顺铂的敏感度,逆转了CNE2/DDP对顺铂的耐药性。

Objective To investigate drug-resistant effect in a cisplatin-resistant human nasopharyngeal carcinoma（NPC） cell line CNE2/DDP with HIF-1α silencing.Methods The drug-resistant cell line CNE2/DDP was established by a procedure of the human NPC cell line CNE2 exposed to the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin.Small fragments of HIF-1α RNA were transfected into the cell line CNE2/DDP to silence HIF-1α by transient transfection method.Cell proliferation and apoptosis of cell line CNE2/DDP with HIF-1α silencing were measured by MTS assay and flow cytometry analysis,respectively.Western blot were applied to detect the expression of HIF-1α and MDR1 in cell line CNE2/DDP.Results The cisplatin-resistant human NPC cell line CNE2/DDP was established successfully and the resistance index was 16.When the concentration of cisplatin was higher than or equal to 1.0 μg/ml,the inhibition rates of cisplatin on CNE2 group and siHIF-1α siRNA group were significantly higher than that on CNE2/DDP group and siCtrl-siRNA group（P〈0.05）.Similarly,flow cytometry analysis showed that apoptosis in CNE2 group and siHIF-1α siRNA group were significantly higher than that in CNE2/DDP group and siCtrl-siRNA group（P〈0.05）.Furthermore,Western blot showed that HIF-1α and MDR1 proteins in siHIF-1α siRNA group were significantly lower than those in CNE2/DDP group.Conclusion HIF-1α silenced by siRNA improved the sensitivity of the cisplatin-resistant human NPC cell line CNE2/DDP to cisplatin,and reversed the resistance of CNE2/DDP to cisplatin.