Reversal Of Multidrug Resistance By RNA Interference

Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. Clinically, MDR is one of the major causes for chemotherapeutic treatment failure in cancer patients. P-gp is a transmembrane glycoprotein capable of transporting a variety of structurally and functionally diverse chemotherapeutic drugs, leading to reduced intracellular drug concentration and decreased cytotoxicity. P-gp antagonists such as cyclosporin A, Verapamil could reverse MDR; however , these drugs often display serious toxicities that limit their clinical use. Directed blocking of MDR1 gene , inhibiting the p-gp-encoding mRNA and p-gp expression , reversing MDR and restoring drug sensitivity to drug-resistant human cancer has been in great demand.The strategies of gene therapies for reversing MDR mainly included antisenseologodeoxyribonucleotides (ASODN)n hammerhead ribozymes and antisense RNA. Since the advent of RNAi approach, RNAi has been widely used both in the area of virology and that of oncology.As a novel and powerful technique for gene silencing ,it was not only been used for gene function analysing ,but also for gene therapy.RNA interference suppress gene expression in mammalian cells through catalytic degradation of mRNAs triggered by short double-stranded RNAs which are combined with the sense and antisense chains on the targeted mRNA ,mediated by amultiprotein cytoplasmic complex. Small interfering RNAs (siRNAs) of approximately 21-23bp mediate sequence specific mRNA degradation in mammalian cells.Objective :Reversal effects of multidrug resistance by RNA interference were studied.We evaluated the efficiency of chemically synthesized mdr1-siRNAs and plasmid-based hairpin mdr1-siRNAs, and investigated the effectiveness of blocking mdr1 gene in multidrug-resistant carcinoma cell lines transfected with chemically synthesized mdr1-siRNAs and hairpin siRNA vectors. Also ,we compared the effectiveness of the anti- mdr1 asodns alone and in combination with mdr1-siRNAs , which was synthesized based on the same sequences.The study included two parts:Part 1: Reversal of multidrug resistance by chemically synthesized siRNAs. SiRNAs and asODNs targing on the same MDR-1 gene sequences were chemically synthesized, withnegative-siRNAs as controls. mdr1-siRNAs and asODNs were transfected into doxorubicin- resistance human leukemia cell lines K652/A02 and breast cancer cell lines MCF-7/ADR by lipofectAMINE2000.The expression of mdr1-RNA and protein was detected by RT-PCR and Western Blott. Rhodamine 123 efflus and Cytotoxicity were tested by flow cytometry and MTT assay. The results showed that siRNAs and adODNs can inhibit P-gp expression ,both at the levels of mdr1-RNA and protein, increase the transporting function of P-gp and restore doxorubicin sensitivity to drug-resistant human cancer cell lines.The reversal effect of low concentration siRNAs(200nmol/L) was greater than that of high concentration (5μmol/L) asODNs .Furthermore,the reversal effort of siRNAs combined with asODNs was better that of siRNAs or asODNs alone (p<0.05 ) .Part 2: Reversal of multidrug resistance by siRNA expression plasmids. We constructed mdr1 siRNA-containing expression plasmids pRNAT-U6.1/Neo-mdr1. The vector carries a Neomycin resistance gene as the selectable marker for establishing stable cell line . A GFP marker (GFP) under CMV promoter control was used to track the transfection efficiency. It used U6 promoter for siRNA expression and carried the insert with BamHI at the 5' end and HindIII at the 3' end.After identification by DNA sequencing,the positive clone was selected and cultured.pRNAT-U6.1/Neo—mdr1 was transfected into breast cancer cell lines MCF-7/ADR by lipofectAMINE2000. After 3 days the expression of GFP was observed by fluorescence microcope and the transfection rate was detected by flow cytometry. After selection by G418 for 3 weeks,the survival cells were cultured for the detection of reversal effort. The mdr 1 mRNA and protein were detected by RT-PCR and Western Blot. Rhodamine 123 efflus and cytotoxicity were tested by flow cytometry and MTT assay. T-test and analysis of variance was used to evaluate the difference between two groups.72h after transfecting , 1 month and 2month after selected by G418 , mdr1 gene can be effectively inhibited, and that the sensitivity to P-gp-transportable drugs can be restored.Conclusions: 1. Multidrug resistance can be reversed by chemically synthesized siRNAs. The reversal effect of low concentration siRNAs(200nmol/L) was greater than that of high concentration (5μmol/L) asODNs .Furthermore,the reversal effort of siRNAs combined with asODNs was better than that of siRNAs or asODNs alone.2. mdr1-siRNA expression plasmids can reverse MDR stably and permanently.