Co-suppression Of Mdr1 And GCS Gene Reverses Multidrug Resistence In Breast Cancer By RNA Interference

【Background】Breast cancer is one of the most common malignant tumor of women.In addition to surgical treatment,chemotherapy and endocrine therapy are important tools in breast cancer treatment.However,multidrug resistance(MDR) to chemotherapeutic agents is a major obstacle to successful chemotherapy in patients with breast cancer. The so-called multidrug resistance refers to that when a kind of tumor become resistent to one drug,it will also become resistent to other drugs which have different structure and mechanisms.The mechanism of MDR in breast cancer is complex,while overexpression of the members of the adenosine triphosphate(ATP)-binding cassette(ABC) membrane transporter family are one of the most important factors.P-glycoprotein(P-gp),coded by multidrug resistance gene-1(mdrl),as other members of ABC membrane transporter family,acts as a drug efflux pump,lowers intracellular drug levels to sublethal concentrations and helps cells to escape from death.It has been reported that notably natural product anticancer agents,including vinca alkaloids,anthracyclines (daunorubicin,adriamycin),and taxanes are the substrates of P-gp and P-gp covers 50%of MDR in breast cancer.So reversing MDR mediated by P-gp will play an important role in the therapy of breast cancer.In order to reverse MDR mediated by P-gp,a number of methods were adopted, including chemical treatment,natural drug therapy,immunology treatment and gene therapy.Chemical synthesis of inhibitors can increase tumor cell sensitivity to chemotherapeutic drugs.However,chemical drugs at therapeutic concentrations often have greater toxicity.At present,it is thought that inhibiting resistance protein mRNA expression selectively may be an effective stragedy to reverse drug resistance.Our group once applicated antisense oligonucleotide as well as ribozyme to reverse P-gp-mediated MDR and the methods were able to inhibit P-gp at different levels,but failed to achieve the desired results.This may be related to the following factors:First, ribozyme has less efficient inhibition of target gene;Second,the degradation of ribozyme and antisense oligonucleotides were limited by the secondary structure of target gene mRNA,so it is difficult to select efficient sequence;Third,drug-resistant tumor cells exist other mechanisms.Recent study reveals that glucosylceramide synthase(GCS) may be one of the factors which could cause tumor multidrug resistance.Glucosylceramide synthase (GCS) allows cells to escape from ceramide-induced cellular apoptosis by mediating ceramide glycosylation.Some researches revealed that accumulation of glucosylceramide(GC),the product of the glycosylation event,is a characteristic of some multidrug-resistant cancer cells and tumors derived from patients who are less responsive to chemotherapy.Many chemotherapy drugs can inhibit the activity of GCS and to increase intracellular ceramide synthesisRNA interference is a new and effective research tool for gene function analysis by introducing double-stranded RNA into cells and leading to the sequence-specific destruction.RNAi can be induced by double stranded RNA(dsRNA),which is cleaved into 21-23bp RNA fragment,knowm as small interference RNA(siRNA),by an RnaseⅢ-like enzyme,Dicer.Then,siRNA is incorporated into a protein complex called RNA-induced silencing complex(RISC) which recognizes and cleaves mRNA in a sequence-specific manner.Because RNAi has the specificity and efficiency,and compared to antisense oligonucleotide,siRNA-mediated gene silencing is more effective and longer duration,we planned to co-suppress the mdrl and GCS gene by plasmids with RNA interference sequences to improve the efficiency of MDR reverse in breast cancer.【Methods】1.The siRNA sequence targeted to mdrl or GCS were designed and inserted into plasmid vector pSUPER,then confirmed by enzyme digestion and DNA sequencing. After that,RNA interference sequence and H1 promoter sequence were obtained by digesting the plasmids by appropriate restriction enzymes and then inserted into pEGFP-C1 with Enhanced Green Fluorescence Protein(EGFP) and stable selective marker.After comfirmed by enzyme digestion and DNA sequencing,the plasmids were amplified.2.Co-transfection of the plasmids with RNA interference sequences into the cell lines MCF-7/ADM which over-expression P-gp and GCS,meanwhile,the blank control group and only a kind of interference plasmid transfection group were set up, respecively.After another 48h culture,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,western blot was used to analysis the P-gp and GCS protein.The function of P-gp was evaluated by Rhodamine 123 (Rh123) effiux assay.MTT method was performed to evaluate the 50%inhibition of concertration of adriamycin and vincristine.3.After transfection with different vectors,G418 was used to select stable expression cell lines.And after 2 weeks,cell lines which stably express RNA interference sequences were established.After cultured for another 3 weeks,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,western blot was used to analysis the P-gp and GCS protein.The function of P-gp was evaluated by Rhodamine 123(Rh123) efflux assay.MTT method was performed to evaluate the 50%inhibition of concertration of adrimaycin and vincristine.Through the above experiments,the efficiency of the long-term reversal by RNA interference was evaluated.【Results】1.After transfecion for 48h,RT-PCR was adopted to detected the expression of mdrl mRNA and GCS mRNA,and the result demonstrated that compared with MCF-7/ADM cells(we considered the relative mRNA levels in MCF-7/ADM cells as 100%),the relative expression of mdrl mRNA in G0,M0 and GM0 decreased to 1.105±0.99%,38.75±9.7%and 0.4491±0.22%,respectively,and that of GCS mRNA decreased to 7.464±3.0%,38.34±16%and 3.142±0.64%,respectively,significantly lower than that of the controls(P<0.01).From further analysis,we found that the mRNA levels in the cells GM0 were much lower than that of G0 or M0(P<0.05).The results of western blot showed that the levels of the two proteins were significantly decreased in the cells GM0 and that the levels of the two proteins in GM0 cells were signifiantly lower than that in G0 or M0 cells.Rh123 effiux test indicated that the fluoresence intensity of the resistence cell lines were 2.727%±0.4834,and that of G0 or M0 were 33.71%±2.267 and 17.25%±1.024,while that in GM0 were 50.12±6.349. Compared to that in the resistence cell lines,the function of P-gp in GM0 down-regulated significantly.MTT method showed that the drug resistence index of G0,M0 and GM0 to adriamycin and vincristine declined significantly,while that of GM0 was also much lower than that of G0 and M0.Furthermore,down-regulated of mdrl could induce decrease of GCS while inhibition of GCS also induced suppression of mdrl.2.After stable cell lines were seleced,RT-PCR was used to detect the expression of mdrl mRNA and GCS mRNA,and the result demonstrated that the relative expression of mdrl mRNA in G1,M1 and GM1 decreased to 46.04±3.8%,13.39±0.69%and 0.6715±0.71%,respectively,and that of GCS mRNA decreased to 64.11±2.7%,25.28±5.9%and 4.321±2.7%respectively,significantly lower than that in MCF-7/ADM(P<0.01).From further analysis,we found that the mRNA levels in the cells GM1 were much lower than that of G1 or M1(P<0.05),while that had no significant differences with the corresponding transient transfection groups.The results of western blot showed that the levels of the two proteins were significantly decreased in the cells GM1 and that the levels of the two proteins in GM1 cells were signifiantly lower than that in G1 or M1 cells.Rh123 efflux test indicated that the fluorescence intensity of G1,M1 and GM1 cell lines all decreased compared to that of MCF-7/ADM.Compared to that in the resistence cell lines,the function of P-gp in RNAi group down-regulated significantly.MTT method displayed that the drug resistence of G1,M1 and GM1 to adriamycin and vincristine declined significantly and there were no significant differences between the stable transfection groups and the corresponding transient -transfection cells.【Conclusions】1.RNA interference could significantly decrease the production of protein by inhibition the expression of mRNA.2.The result of transient transfection demonstrated that suppression of mdrl or GCS gene could efficiently reverse MDR in breast cancer,while co-suprression of both mdrl and GCS was more efficient than inhibiting alone.3.The result of stable transfection showed that the plasmids with RNA interference sequences could inhibit the expression of target gene for a long period(at least for 3 weeks).4.The result of the experiment indicated that down-regulated of mdrl could induce decrease of GCS while inhibition of GCS also induced suppression of mdrl which revealed that mdrl and GCS could affect each other in breast cancer.