RNAi逆转肾癌细胞MDR1基因表达导致的替尼泊苷耐药  

Suppression of MDR1 gene expression and reversal of teniposide resistance in renal carcinoma cell by RNA interference fragment

作　　者：郝怡鑫[1] 贺铮雯[2] 朱建华[1] 沈茜[2] 李秋文[1] 肖文华[1] 

机构地区：[1]解放军总医院第一附属医院肿瘤一科,北京100037 [2]第二军医大学附属长海医院实验诊断科,上海200433

出　　处：《海南医学院学报》2014年第2期149-153,共5页Journal of Hainan Medical University

基　　金：解放军总医院苗圃基金(10KMM35)~~

摘　　要：目的:研究RNA干扰(RNA interference,RNAi)对肾癌细胞多药耐药基因(MDRl)表达的抑制作用,并分析干扰前后肾癌细胞对替尼泊苷(VM-26)敏感性的变化。方法:根据MDRl基因设计3条小干扰RNA(small interfering RNA,siRNA)序列,在质粒的介导下转染肾癌A498细胞,RT-PCR法分析转染前后MDR1mRNA表达水平,筛选出干扰效率最高的siRNA序列;进而利用慢病毒包装siRNA重组质粒,感染A498细胞,RT-PCR法筛选沉默效果最好的细胞株进行克隆,Western bolt法检测MDR1蛋白表达水平,MTT法对比干扰前后替尼泊苷对细胞半抑制浓度(IC50)的变化。结果:3条siRNA序列均能不同程度地抑制细胞MDR1基因的表达,其中siRNA-1序列能更有效地封闭MDRl基因,使MDRl mRNA表达水平下降67%;筛选出的稳转细胞株与未干扰细胞株相比,MDR1蛋白表达量明显下降,并使替尼泊苷对细胞的IC50降低约79.8%,差异具有统计学意义(P<0.01)。结论:RNAi可抑制肾癌A498细胞MDRl基因的表达从而逆转其对替尼泊苷的耐药,使肾癌细胞化疗耐药逆转成为可能。Objective.- To investigate the effect of RNA interference （RNAi） fragment on the ex- pression of MI）R1 gene and the reversal of teniposide（VM 26） resistance in human renal carcinoma cell line. Methods. Three small interfering RNA（siRNA） sequence targeting MDRI gene were synthesized and transfected into renal carcinoma A498 cell, the expression level of MDR1 mRNA was assayed to identify the most effective siRNA fragment using RT-PCR method. After the A498 cell was infected by MDR1-RNAi lentivirus fluid,the optimal gene silencing MDRI-RNAi A498 cell was identified by RT-PCR method. Af- ter the cell was cloned, the MDR1 protein expression level was verified by Western blot method. The ICS0

关 键 词：RNA干扰 小干扰RNA 多药耐药基因 替尼泊苷 

分 类 号：R737.11[医药卫生—肿瘤]