RNAi-based Knockdown of Multidrug Resistance-associated Protein 1 is Sufficient to Reverse Multidrug Resistance of Human Lung Cells

Abstract

Up-regulation of multidrug resistance-associated protein 1 (MRP1) is regarded as one of the main causes formultidrug resistance (MDR) of tumor cells, leading to failure of chemotherapy-based treatment for a multitude ofcancers. However, whether silencing the overexpressed MRP1 is sufficient to reverse MDR has yet to be validated.This study demonstrated that RNAi-based knockdown of MRP1 reversed the increased efflux ability and MDRefficiently. Two different short haipin RNAs (shRNAs) targeting MRP1 were designed and inserted into pSilence-2.1-neo. The shRNA recombinant plasmids were transfected into cis-dichlorodiamineplatinum-resistant A549lung (A549/DDP) cells, and then shRNA expressing cell clones were collected and maintained. Real time PCRand immunofluorescence staining for MRP1 revealed a high silent efficiency of these two shRNAs. Functionally,shRNA-expressing cells showed increased rhodamine 123 retention in A549/DDP cells, indicating reduced effluxability of tumor cells in the absence of MRP1. Consistently, MRP1-silent cells exhibited decreased resistance to3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and DDP, suggesting reversal of MDRin these tumor cells. Specifically, MRP1 knockdown increased the DDP-induced apoptosis of A549/DDP cellsby increased trapping of their cell cycling in the G2 stage. Taken together, this study demonstrated that RNAibasedsilencing of MRP1 is sufficient to reverse MDR in tumor cells, shedding light on possible novel clinicaltreatment of cancers.