小干扰RNA沉默MDRl基因对SMMC-7721肝癌细胞株多药耐药性的影响

Silencing MDRI gene by siRNA enhanced sensitivity to chemotherapy of human hepatoma cells

目的将表达特异性的小干扰RNA（siRNA）导入肝癌耐药细胞，干扰多药耐药（MDRl）基因后，观察其对肝癌耐药细胞化疗敏感性的影响。方法使用浓度梯度法制作sMMC-7721耐阿霉素细胞株（SMMC-7721／ADM），阿霉素浓度从0．1～2．0ms／L；转染siRNA（浓度25、50、75nmo/L）沉默此耐药株MDRl基因；转染后24、48、72h用定量逆转录．聚合酶链反应（qRT．PCR）检测各组细胞MDRl的mRNA表达水平；siRNA（浓度50nmoVL）转染后48h，行噻唑蓝（MTT）比色法检测细胞对化疗药物的敏感性，计算各药物的半数抑制浓度（IC50）和耐药系数（RI）；流式细胞仪（FCM）检测细胞对阿霉素（浓度0．2mg／L）的凋亡。Westernblot检测蛋白表达含量。结果SMMC-7721／ADM对阿霉素、5．氟尿嘧啶（5．Fu）、长春新碱、奥沙利铂的RI为25．43、68．32、39．17、18．31。siRNA转染沉默MDRl基因后，发现其对上述药物的RI为5．01、17．04、4．96、1．62；qRT．PCR发现其MDRlmRNA表达量显著降低（P〈0．05）；Westernblot显示其MDRl表达产物P—gp蛋白显著降低（P〈0．05）；FCM发现加入阿霉素后，其细胞凋亡指数显著大于沉默前（P〈0．05）。结论用siRNA沉默SMMC-7721／ADM的MDRl基因后，SMMC-7721／ADM对化疗药的敏感性显著增加。MDRl基因与SMMC-7721肝癌细胞的多药耐药性显著相关，可通过沉默其MDRl基因来提高耐药肿瘤细胞对化疗药物的敏感性。

Objective Hepatocellular carcinoma （HCC） responds poorly to chemotherapy owing to multidrug resistance （MDR）. This study was designed to observe the effect of small interfering RNA （siRNA） targeting MDR1 in modulating drug resistance and apoptosis. Methods HCC MDR cell lines, SMMC-77Tl/adriamycin （ADM）, were developed by exposing parental cells to stepwise increasing concen- trations of ADM from 0. 1 to 2.0 mg/L. After siRNA （25, 50, 75 nmol/L） transfection MDR1 mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） at 24, 48, 72 h. methyl thiazol tetrazolium （MTT） assay was used to determine drug sensi- tivity 48 h after transfection. Flow cytometry （FCM） was employed to analyze cell apoptosis to ADM （0. 2 mg/L）. The expression of MDR1 protein was examined by Western blotting. Results MTT assay showed that the resistance index （RI） values of SMMC-7721/ADM cells against ADM, 5-Fu, VCR and oxaliplatin were 25.43, 68. 32, 39.17, 18. 31, and those after siRNA transfection were 5.01, 17.04, 4. 96, 1.62, respectively. QRT-PCR analysis demonstrated the MDR1 mRNA expression was decreased significantly in SMMC-7721/ADM cells after siRNA transfection （ P 〈 0. 05 ）. In addition, as compared with parental cells, MDR1 protein expression was apparently decreased in SMMC7721/ADM cells treated with siRNA （ P 〈 0.05）. Flow cytometry revealed significant apoptosis of the cells following siRNA transfection （ P 〈 0. 05）. Conclusion Inhibition of MDR1 by siRNA enhanced the selectively restored sensitivity to drugs. The MDR1 siRNA might represent a new therapeutic option for HCC.