siRNA介导多药耐药相关蛋白(MRP的表达沉默(英文)

Silencing of Multidrug Resistance-Associated Protein (MRP1) Expression by siRNAs

利用pSIREN-RetroQ载体构建了3个沉默多药耐药相关蛋白(MRP1)基因表达质粒pSIREN-siRNAs.并通过限制性内切酶酶切鉴定和DNA测序鉴定,将截断的MRP1和全长MRP1cDNA分别克隆到真核表达载体pEGFP-N2和pcDNA3.1中,产生了pEGFP-MRP1T和pcDNA-MRP1表达质粒.质粒pEGFP-MRP1T分别与3个pSIREN-siRNAs共转染HEK293细胞沉默MRP1T-GFP靶基因,pSIREN-siRNA1作为阴性对照.荧光显微镜下显示结果表明,与pSIREN-siRNA1相比,pSIREN-siRNA2和pSIREN-siRNA3产生的siRNA能够有效沉默MRP1T-GFP融合蛋白的表达.为了沉默全长MRP1基因的表达,pcDNA-MRP1分别与3个pSIREN-siRNAs共转染HEK293细胞.Western印迹和MTT分析表明,pSIREN-siRNA2和pSIREN-siRNA3能有效抑制190kDMRP1在HEK293细胞中的表达,而pSIREN-siRNA1则不能.pSIREN-siRNA2和pSIREN-siRNA3能逆转MRP1转染HEK293细胞产生的多药耐药性.RNA二级结构预测结果分析表明,siRNA1靶序列mRNA局部自由能热动力参数ΔG低于siRNA2和siRNA3靶序列mRNA局部自由能热动力参数,siRNA1的GC含量和Tm值高于siRNA2和siRNA3.这些数据提示,siRNA和局部靶结构可能影响siRNA对MRP1 mRNA表达的沉默作用.

Three pSIREN-siRNA plasmids were constructed using a pSIREN-RetroQ vector to silence the expression of multidrug resistance-associated protein （MRP1） gene, and subsequently characterized by restriction endonuclease digestion and DNA sequencing. A truncated MRP1 and a full-length MRP1 were cloned into pEGFP-N2 and pcDNA3. 1 respectively as pEGFP-MRP1T and pcDNA-MRP1. The plasmid pEGFP-MRP1T was co-transferred with each of the three pSIREN-siRNAs into HEK293 cells for MRP1 T-GFP targeted silencing, and pSIREN-siRNA1 was used as the negative control, pSIREN-siRNA2 and pSIREN-siRNA3 appeared to be more effective to silence MRP1T-GFP compared to pSIREN-siRNA1 as shown by fluorescence microscopy. For the silencing of full-length MRP1 expression, HEK293 ceils were co-transferred with pcDNA-MRP1 and either of the three pSIREN-siRNAs, then subjected for Western blot analysis and MTT assays, pSIREN-siRNA2 and pSIREN-siRNA3 were able to inhibit the expression of 190 kD MRP1, but not pSIREN-siRNA1. The MDR of MRPl-transfected HEK293 cells was abolished with pSIREN-siRNA2 or pSIREN-siRNA3 transfections. RNA secondary structure predictions demonstrated that the mRNA local free energy （AG） of the siRNA1 targeted sequence was lower, as the GC content and Tm value of siRNA1 were higher than those of siRNA2 and siRNA3. These data suggest that the local structure siRNAs and target mRNA may influence the silencing efficiency of MRP1 expression.