RNA干扰抑制MDR1表达并逆转Bel7402/5-Fu肝癌细胞耐药性的研究

Suppression of MDR1 expression and restoration of sensitivity to chemotherapy in multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu by RNA interference

目的:研究小片段RNA干扰对肝癌耐药细胞系Bel7402/5-Fu中多药耐药基因(multidrugresistance1,MDR1)及其蛋白产物P-gp表达的抑制作用和逆转其耐药性的效果。方法:合成针对MDR1启动子区域的RNA干扰小片段,转染肝癌耐药细胞Bel7402/5-Fu。通过RT-PCR和Westernblot方法,在mRNA和蛋白水平评价RNA干扰对MDR1表达的影响。MTT法检测RNA干扰逆转Bel7402/5-Fu细胞耐药性的效果,按实验组和对照组细胞的半数抑制剂量(IC50)计算RNA干扰对细胞耐药倍数的改变和RNA干扰组细胞的耐药逆转倍数。以流式细胞仪比较各组细胞在相同浓度化疗药物作用时细胞的凋亡情况。结果:在耐药肝癌细胞Bel7402/5-Fu中,RNA干扰明显抑制了MDR1mRNA和蛋白产物P-gp的表达水平,其表达仅为对照组细胞的22.55%和25.49%(P<0.01)。在相同浓度化疗药物的作用下,RNA干扰组Bel7402/5-Fu细胞凋亡比例显著高于对照组(P<0.01),表明细胞耐药性显著下降,对5-Fu的耐药逆转倍数为14.88倍。结论:肝癌耐药细胞Bel7402/5-Fu中,RNA干扰对MDR1mRNA及其蛋白产物P-gp的表达水平有显著抑制作用,具有良好的逆转耐药性的效果。

Objective To investigate the suppression of MDR1 and P-glycoprotein induced by small interfering RNA and the restoration of sensitivity to chemotherapeutic drugs in multidrug-resistant hepatoeellular carcinoma cell line Be17402/5-Fu. Methods MDR1-targeted small interfering RNA duplexes were introduced into multidrug-resistant hepatocellular carcinoma cell line Be17402/5-Fu. The suppression of MDR1 and its gene product P-glycoprotein was examined by RT-PCR and Western blot. MTT assay was performed to measure the reverse effect of small interfering RNA based on the results of IC50. Cell apoptosis was assessed by flow cytometry after various cell lines were treated with chemotherapeutic drugs. Results The overexpression of MDR1 and P-glycoprotein was suppressed efficiently by the introduction of small interfering RNA, which caused sequence-specific gene silence. The level of MDR1 in the transfected Be17402/5-Fu cells reduced to 22.55% and P-glycoprotein to 25.49% compared with those of the controls. The apoptosis rate of Be17402/5-Fu cells increased significantly in the siRNA group during the chemotherapy （ P 〈 0. 01 ）. Their resistance to 5-Fu was reversed by 14.88 folds, which indicated the restoration of sensitivity to drugs. Conclusion Small interfering RNA can inhibit MDR1 expression effective and reverse the multidrug resistance mediated by P-glycoprotein.