靶向肿瘤多药耐药基因mdr1的siRNA的构建与鉴定

Abstract

[中文文摘]mdr1基因及其表达产物P-gp是引起肿瘤细胞多药耐药(MDR)的主要原因,抑制mdr1基因的表达可用于逆转MDR.RNAi可用于特异抑制靶基因的表达,本研究的目的是构建获得可特异有效靶向mdr1基因的siRNA元件.应用siRNA设计软件与mRNA结构分析软件设计构建了3个分别靶向mdr1基因mRNA环结构和茎结构的siRNA元件,同时构建了携带mdr1基因序列的luc报告质粒,通过siRNA表达质粒与携带靶序列的报告质粒的共转染抑制实验检测不同siRNA的抑制效率,结果显示靶向环结构siMDR1B具有较好的抑制效率和特异性.进一步将siMDR1B表达载体与mdr1基因表达载体共转染细胞,应用免疫流式细胞术检测显示,相比对照细胞,siMDR1B可显著抑制其转染后mdr1基因产物P-gp蛋白的表达活性.同时采用CCK-8细胞活性检测试剂评价了siMDR1B对细胞活性的影响,结果显示siMDR1B不会影响细胞活性,具有良好的特异性.本研究获得的可有效靶向mdr1基因的siRNA元件可为进一步开展逆转MDR研究提供重要基础.[英文文摘]The over-expression of mdr1 gene is the main reason of multidrug resistance(MDR) of tumor cells.Inhibiting the expression of mdr1 gene can be used to reverse the MDR.RNA interfence(RNAi) is a sequence-specific intracellular mechanism of gene silence.In this study,the construction of small interfering RNAs(siRNAs) element against mdr1 gene effectively and specifically were explored.Three siRNA sequences targeting to mRNA stem or loop stucture separately were selected based on the sequence of mdr1 gene.Using various reporter plasmids contaiining different target sequences,the inhibition efficiencies and specificities of the constructs against mdrlgene were determined and siMDR1Btargeting to the loop structure was selected from the candidates.siMDR1B expression vector was then co-transfected with the mdrigene expression vector in cells,and the expression level of P-gp protein encoded by mdr1gene was analysed by immune flow cytometry.Results showed that P-gp protein expression can be effectively inhibited by siMDR1Bin transformed cells comparing to control cells.CCK-8assay was used for cell viablity analsis in this study,and results showed that cell viability was not affected by siMDR1Bexpression in transformed cells.In conclusion,siMDR1Bis a promising candidate for the next research on MDR reverse.