小干扰RNA沉默多药耐药相关蛋白基因2对人肝癌细胞HepG2多药耐药的逆转作用  

Silencing multidrug resistance-associated proteins 2 gene by siRNA reversed multidrug resistance in hepatocellular carcinoma cells

作　　者：褚忠华[1] 张大伟[1] 刘建平[1] 韦金星[1] 苏正[1] 陕基德[1] 

机构地区：[1]中山大学孙逸仙纪念医院肝胆外科,广州510120

出　　处：《中华实验外科杂志》2012年第8期1547-1549,共3页Chinese Journal of Experimental Surgery

基　　金：广东省科技计划资助项目（20088080703054）

摘　　要：目的检测小干扰RNA（siRNA）对肝癌耐药细胞株HepG2／阿霉素（ADM）多药耐药相关蛋白基因2（MRP2）及其蛋白产物的抑制作用，观察对多药耐药性的影响。方法使用浓度梯度法制作HepG2耐ADM细胞株（HepG2／ADM），ADM浓度从0．1～2．0mg／L；设计合成针对MRP2的siRNA小片段，转染HepG2／ADM耐药细胞，24h后通过实时荧光定量逆转录一聚合酶链反应（RT—qPCR）和Westernblot检测siRNA对MRP2基因mRNA和蛋白的抑制效果；噻唑蓝（MTT）比色法观察抑制MRP2基因表达后，HepG2／ADM细胞对化疗药物的敏感性变化，并计算各药物的半数抑制浓度（IC50）。结果HepG2／ADM对ADM、5-氟尿嘧啶（5-Fu）、长春新碱和奥沙利铂的IC50值分别为0．3204、3．8002、0．2014和0．1221；siRNA明显抑制了HepG2／ADM细胞MRP2基因mRNA和蛋白的表达（P〈0．05）；转染MRP2-siRNA后，HepG2／ADM细胞对ADM、5-Fu、长春新碱和奥沙利铂的IC50值明显减小，分别为0．1023、1．4417、0．0452和0．0268。结论用siRNA沉默MRP2基因能够逆转HepG2／ADM细胞对化疗药物的耐药性，MRP2基因与HepG2／ADM肝癌细胞的多药耐药性相关，可通过沉默MRP2基因提高耐药细胞对化疗药物的敏感性。Objective To investigate the suppression of multidrug resistance-associated proteins 2 （MRP2） and the protein product induced by small interfering RNA （siRNA） in the muhidrug-resistant （MDR） hepatocellular carcinoma （HCC） cell line HepG2/adriamyciu （ADM） and the effect on MDR. Methods MDR HepG2/ADM cells were developed by exposing parental cells to stepwise increasing con- centrations of ADM from 0. 1 to 2.0 mg/L. MRP2 targeted small interfering RNA fragments were designed and synthesized, and transfeeted into MDR HepG2/ADM cells. The suppression of MRP2 and the protein product were detected by using real-time reverse transcription quantitative polymerase chain reaction （RT-qPCR） and Western blotting at 24 h after transfcction. Methyl thiazol tetrazolium （MTT） assay was used to determine drug sensitivity of HepG2/ADM cells before and after transfeetion based on the results of ICs0. Results MTI＇ assay showed that the IC50 values of HepG2/ADM against ADM, 5-fluorouracil, vin- cristine, and oxaliplatin were 0. 3204, 3. 8002, 0. 2014 and 0. 1221 respectively, siRNA trausfection sig- nificantly inhibited the expression of MRP2 mRNA and protein in HepG2/ADM cells （ P 〈 0.05 ）. After MRP2-siRNA transfeetion, the ICs0 values of HepG2/ADM against ADM, 5-fluorouraeil, vincristine, and ox- aliplatin were decreased significantly （0. 1023, 1. 4417, 0. 0452 and 0. 0268, respectively）. Conclusion Silencing MRP2 genes by siRNA can reverse MDR of HepG2/ADM cells. MRP2 is closely related to MDR of HepG2/ADM HCC cells and silencing MRP2 might improve the sensitivity of resistant cells to chemo- therapeutic drugs.

关 键 词：癌 肝细胞 多药耐药性 多药耐药相关蛋白基因2 小干扰RNA 

分 类 号：R735.7[医药卫生—肿瘤]