Invitro And In Vivo Inhibitioon Of Mrp Gene Expression And Reversal Of Multidrug Resistance By Sirna

Background and Purpose: MDR (multi-drug resistance, MDR) is one of the majorcauses of failures in human cancer chemotherapy. Many reasons cause MDR, amongwhich overexpression of MRP is a major reason. RNAi is a process which can causegene knock down in sequence specific manner. In this study, we studied thecytotoxicity effect of antineoplastics to three human tumor cells and nude micetumor xenografts after transfecting siRNAs and try to find a novel way to overcomeMDR in cancer therapy. Methods: Four different MRP1-targeted siRNA duplexeswere designed and synthesized by Dharmacon. In vitro, NCI-H460, U251, MCF-7cells in exponential phase of growth were transfected with the four siRNA duplexesas directed by the manufacturer protocol for lipofectamineTM2000-based transfection.Silencing was examined 24 and 48 hours after transfection by reverse transcription-PCR (RT-PCR). Two duplexes which had the better inhibiting effect were thentransfected the three cell lines to investigate the change of tumor cells sensitivity tocytotoxic agents including Epirubicin, Vincristine and Taxinol. The inhibition ofMRP1 protein was examined by Western Blot 72 hours after transfection and thesuppression of cell growth was evaluated by the MTT method.As a result, oneduplex which had the best silencing effect of reversing MDR among the fourduplexes in vitro was chosen. In vivo, to test the cytotoxicity effct of Epirubicinwhen co-administration of siRNA, tumor growth suppression experiments wereconducted in nude mice bearing human NCI-H460 solid tumors. Tumor xenograftswere transfected with MRP1 siRNA by electroporation combinated with intro-tumor injection method. Silence was examined 24, 48, 72houes after transfection by RTPCRand immunohistochemistry. To further determine whether introduction ofMRP1 siRNA could increase the inhibition of Epibubicin to tumor xenogrsfts, micewere transfcted with siRNA twice in the process of chemotherapy. The curve oftumor growth was drawn according to tumor volume. In the end of the research, thetumor tissue was excised, weighted and calculated inhibition rate. ResultsResults: 24 and48 hours after transfecting to NCI-H460, U251 and MCF-7 cell lines, the results ofRT-PCR showed that the four siRNA duplexes significantly reduced the levels of theMRP1 mRNA and siRNA2 and siRNA4 had the better silencing effect. WhensiRNA2 and siRNA4 were used in the following research, the protein of MRP1 in thethree cell lines were significantly decreased 72 hours after transfection as determinedby Western blot and the MDR of cells to Epirubicin and Vincristine were reverseddetermined by MTT method. siRNA2 was proved to have the better silencing effet invitro. In vivo, the results of RT-PCR and immunohistochemistry showed that mRNAand protein of MRP1 were inhibited effectively after transfection of siRNA2 in situfollowed by electroporation. When the animals bearing the tumor were coadministratedsiRNA with Epirubicin, the results demonstrated that siRNAtransfection improved the sensitivity of tumors to Epirubicin compared with nontransfectedtumors. Conclusions: Our research showed that transfection of siRNAtargeted MRP1 gene can effectively inhibit the mRNA and protein of MRP, thesensitivity of tumors to chemotherapeutic drugs was improved both in vitro and invivo models. Co-administration of siRNA and chemotherapeutic drugs provides apromising way in cancer therapy.