Silencing of Multidrug Resistance-Associated Protein(MRP1) Expression by siRNAs / 中国生物化学与分子生物学报

ABSTRACT

Three pSIREN-siRNA plasmids were constructed using a pSIREN-RetroQ vector to silence the expression of muhidrug 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 PeDNA3.1 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 MRP1T-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 MRP1-transfected HEK293 ceils was abolished with pSIREN-siRNA2 or pSIREN-siRNA3 transfections. RNA secondary structure predictions demonstrated that the mRNA local free energy (△G) 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.