小干扰RNA调控多药耐药相关蛋白1基因对SMMC7721肝癌细胞株多药耐药性的影响  

Silencing multidrug resistant-associated protein 1 gene by RNA interference enhanced sensitivity to chemotherapy of human hepatoma cells

作　　者：汪洋[1] 褚忠华[2] 刘建平[2] 苏正[2] 张华耀[2] 

机构地区：[1]武警广东省边防总队医院普通外科,深圳518029 [2]中山大学孙逸仙纪念医院肝胆外科

出　　处：《中华实验外科杂志》2013年第9期1889-1892,共4页Chinese Journal of Experimental Surgery

基　　金：广东省科技计划资助项目（2011B031800296）

摘　　要：目的 将表达特异性的小干扰RNA（siRNA）基因片段导入SMMC7721耐药细胞,干扰多药耐药相关蛋白1 （MRPl）基因后,观察其对细胞化疗敏感性的影响.方法 使用浓度递增法制作耐阿霉素（ADM）细胞株,将ADM浓度在2 mg/L时培养出的细胞,命名为SMMC7721/ADM细胞,转染25、50、75 nmol/L siRNA沉默此耐药株MRP1基因,于转染后24、48、72 h使用实时定量逆转录聚合酶链反应（RT-qPCR）检测各组细胞MRPl mRNA表达水平.于12、24、36、48、60 h用噻唑蓝（MTT）法检测细胞对化疗药物的敏感性,加入反应试剂进行流式细胞仪检测细胞凋亡水平,使用Western blot分别检测细胞中MRP1蛋白表达含量.细胞注入裸鼠右侧肩胛区,14 d后测量肿瘤的长径和宽径.结果 SMMC7721/ADM细胞对阿霉素、5-氟尿嘧啶（5-Fu）、长春新碱（VCR）、奥沙利铂（OHP）的耐药系数分别为25.43、68.32、39.17、18.31.RT-qPCR检测沉默效率,可见MRP1的表达明显降低（P＜0.05）.MRP1基因沉默后,Western blot技术可见MRP1蛋白明显降低.流式细胞仪可见细胞凋亡明显多于沉默前（P＜0.05）.多药耐药性的检测可见细胞对上述药物耐药系数降低为5.73、15.86、2.49、1.84.动物实验监测肿瘤生长,可见肿瘤生长率明显降低.结论 SMMC7721耐药细胞株在使用siRNA沉默MRP1基因后,对化疗药的耐药性有明显下降.MRP1基因与SMMC7721肝癌细胞的多药耐药性显著相关,可通过沉默MRP1基因来提高耐药肿瘤细胞对化疗药物的敏感性.Objective To observe the effect of small interfering RNA （siRNA） targeting multidrug resistant-associated protein 1 （MRP1） gene in modulating multidrug drug resistance （MDR） and apoptosis of SMMC7721/ADM cells.Methods MDR was maintained by culturing the cells with 2 mg/L adriamycin （ADM） and MDR cells were named SMMC77212/ADM cells.The cells were transfected with 25,50 and 75 nmol/L siRNA using the reagent following the manufacturer＇ s protocol.MRP1 mRNA expression level was detected by using real-time reverse transcriptase-polymerase chain reaction （RT-qPCR）at 24,48 and 72 h.Total cells were harvested at 12,24,36,48 and 60 h after treatment with different drugs,then methyl thiazol tetrazolium （MTT） assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution,and the expression levels of proteins were analyzed by using Western blotting.In tumorigenicity experiment,SMMC-7721/ADM cells were transduced with or without MRP1 siRNA,and tumors were monitored for length and width at 14th day.Results MTT assay showed that the resistance index values of SMMC7721/ADM cells to ADM,5-Fu,VCR and OHP were 25.43,68.32,39.17 and 18.31,and 5.73,15.86,2.49 and 1.84 before and after treatment with siRNA respectively.RT-qPCR demonstrated the MRP1 mRNA expression was decreased significantly in SMMC7721/ADM cells after siRNA transfection.As compared with the expression of parental cells,MRP1 protein expression was apparently decreased,and flow cytometry showed the increased apoptosis after siRNA transfection.Conclusion Inhibition of MRP1 by siRNA enhanced the selectively restored sensitivity to drugs.The MRP1 siRNA might represent a new therapeutic option for hepatocellular carcinoma.

关 键 词：癌 肝细胞 多药耐药基因 多药耐药相关蛋白1 RNA干扰 

分 类 号：R735.7[医药卫生—肿瘤]