Objective To explore the correlation between Y-box binding protein-1 ( YB-1) and P-glycoprotein ( P-gp) in drug-resistant hepatocellular carcinoma ( HCC) Bel-7402/ADM cells, and speculate the related mechanism of drug resistance.Methods Bel-7402/ADM cells were developed by concentration gradient escalation and intermittent administration of large dose. The levels of YB-1 mRNA and MDR1 mRNA were detected by means of RT-PCR.Western blotting was used to detect the protein expression of YB-1 and P-gp in the Bel-7402 cells and doxorubicin resistant Bel-7402 ( Bel-7402/ADM) cells. Bel-7402/ADM cells were transfected with small interfering RNA ( siRNA) targeting human YB-1. Expression levels of YB-1 and MDR1 mRNA and protein were detected by means of RT-PCR and Western blotting. Results IC50 values of ADM on hepatoma carcinoma cells Bel-7402 and Bel-7402/ADM were (2.23±0.07) and (7.02±0.03) μmol?L-1. The mRNA expression levels of MDR1 and YB-1 were all significantly higher in Bel-7402/ADM cells than in Bel-7402 ( P<0.01) . The mRNA expression levels of MDR1 and YB-1 in Bel-7402/ADM cells transfected with YB-1 siRNA were reduced significantly (P<0.01). The protein levels of YB-1 and MDR1 in Bel-7402/ADM cells transfected with YB-1 siRNA were reduced significantly ( P<0. 05 ) . Conclusion These results suggest that the high expression level of YB-1 is probably correlated with multidrug resistance in HCC Bel-7402/ADM cells.%目的 研究Y-box结合蛋白1(YB-1)与人肝癌耐药细胞Bel-7402/ADM中P糖蛋白(P-gp)的关系,推测其诱导癌细胞产生耐药性的相关机制.方法 以浓度梯度递增联合大剂量间断冲击诱导法建立人肝癌耐药细胞株Bel-7402/ADM?运用实时荧光定量聚合酶链反应(RT-PCR)检测YB-1、P-gp在人肝癌耐药细胞株Bel-7402/ADM和相应敏感株mRNA含量?采用Western blotting检测YB-1、P-gp在两种细胞株的表达量?将YB-1 siRNA转染耐药细胞,检测转染前后YB-1和MDR1在mRNA水平和蛋白水平的变化.结果 多柔比星对Bel-7402和Bel-7402/ADM的半数抑制浓度(IC50)值分别为(2.23±0.07)和(7.02±0.03)μmol?L-1.耐药组MDR1 mRNA的表达量高于亲本细胞(P<0.01),YB-1 mRNA的表达量也显著高于亲本细胞(P<0.01).耐药组P-gp表达量高于亲本细胞(P<0.05),YB-1表达量也显著高于亲本细胞(P<0.01).耐药细胞转染siRNA后YB-1 mRNA、MDR1 mRNA的表达显著降低(P<0.01),YB-1、MDR1蛋白水平较转染前显著降低(均P<0.05).结论 YB-1表达与P-gp表达密切相关,YB-1与人肝癌耐药细胞株Bel-7402/ADM多药耐药相关.
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