Studies On Screening Of SiRNA Of MDR1Gene And Its Reversal Of Multidrug Resistance In Breast Cancer

Objective: The multidrug resistance of tumor is the leading cause ofcancer chemotherapy failure. Multidrug resistance （MDR） is a kind ofresistance to chemotherapeutic drugs as well as to other chemotherapy drugswhich structures and functions are different. P-glycoprotein （P-gp） is encodedby MDR1gene and it is a multidrug resistance-associated protein. It is anATP-dependent drug efflux pump and can expel the chemotherapy drugs fromthe cells, thus leading to the failure of chemotherapy. After chemotherapy,tumor cells can quickly express MDR1gene, and the small interference RNA（siRNA） can cause degradation of the homologous target mRNA, result inspecific silencing of gene expression. Therefore, the use of siRNA to inhibitthe expression of MDR1gene mRNA would be a viable means to reversetumor multidrug resistance. In this study, the siRNA that could effectivelyinhibit the expression of MDR1gene was screened, the siRNA expressionplasmid with U6and H1double promoters was constructed, and the cationicliposomes of siRNA expression plasmid were prepared and used to reversemultidrug resistance in breast cancer cells in combination withchemotherapeutic drugs. Our studies lay the foundation for the research anddevelopment of siRNA drug and gene therapy of multidrug resistance.Methods:（1） Design siRNA sequences:Select the human MDR1gene （serial number NM₀00927） as the target,obtain siRNA candidate sequences using siRNA design software, analyzemRNA secondary structure using biological informatics methods, analyzehomology with BLAST, then determine the final sequences and synthesizedirectly by the biotechnology company.（2） Screen effective siRNA sequences: The siRNAs were transfected into drug-resistant breast cancer cell lineMCF-7/ADR. Quantitatively analyze MDR1gene expression at the mRNAlevel （Real-time PCR） and the protein level （Western Blot） respectively. ThesiRNA sequence with the high inhibition efficiency was screened and used insubsequent experiments.（3） Further test of selected siRNA:MTT experiment was carried out to assay the sensitivity of transfectedcells to adriamycin. Dose-response relationship curve was set up and theeffective concentration range of siRNA was determined through usingreal-time PCR to detect of the suppression of target gene.（4） Construct siRNA expression vector:Chemically synthesize two complementary single stranded DNA thatcorresponds to siRNA sequences, both ends of which has the HindIII andBglII restriction sites. After annealing, linkage of DNA sequences with pDualplasmid that was digested by HindIII and BglII. The ligation product wastransformed into E.coli DH5α and monoclonal colonies were selected toculture on next day. The plasmids were extracted and sequenced.（5） Preparation the cationic liposomes of siRNA expression vector andanalyze its physiochemical properties:The cationic lipid, PEG lipid and cholesterol were dissolved in ethanolwith a certain ratio and the blank liposomes were prepared by ethanolinjection method. The blank liposomes were mixed rapidly with equal volumeof siRNA expression plasmid solution, during which the nucleic acid wasactively loaded into liposomes and nano-sized cationic liposomes of siRNAplasmid were achieved. The properties of liposomes were determinedincluding electric potential, particle size and encapsulation efficiency. Thechemical stability after liposome encapsulation including enzyme stability andserum stability was also determined.（6） Pharmacokinetic study of siRNA plasmid liposomes in mice:The method relying on Realtime PCR was set up to detect siRNAplasmid in plasma, based on which, the concentration of plasmid in blood could be determined and the half-life of plasmid after liposome encapsulationcould be obtained.（7） Pharmacodynamic study of siRNA plasmid liposomes in vitro:Realtime PCR was used to assay the MDR1gene expression levels ofdrug-resistant breast cancer cells MCF-7/ADR after transfected with siRNAplasmid liposomes.SRB was used to assay the sensitivity of cells toadriamycin after transfected with siRNA plasmid liposomes.（8） Pharmacodynamic study of siRNA plasmid liposomes in vivo:Drug-resistant breast cancer model in SCID nude mouse was established.The animals were randomly divided into three groups by tumor volume,including control group, adriamycin group, siRNA plasmid liposomes plusadriamycin group. The drugs were administrated via tail vein injection once aweek. The health status of mice was observed every day. Tumor volume andanimal body weight were determined every other day. After the end of animalexperiment, all mice were sacrificed and tumor was excised. Tumor size wasobserved and tumor weight was used to calculate inhibition rate. The MDR1gene expression levels of tumor were detected using real-time PCR.Results:（1） Design siRNA sequences:The siRNAs directed to MDR1gene were designed based on the siRNAdesign principles. The secondary structure and homology of siRNA wereanalyzed, and eight siRNA sequences were obtained.（2） Screen effective siRNA sequences:Realtime PCR results showed that compared with the control, negativecontrol had no effect, and siRNA4,5,6and8could not induce the silence ofMDR1gene. However, the MDR1expression levels in positive control groupand in the groups transfected with siRNA1,2,3and7reduced63%（P <0.05）,83%（P <0.0001）,68%（P <0.0001）,68%（P <0.01）, and70%（P <0.0001）,indicating that these siRNA sequences could silence the MDR1gene. WesternBlot results showed that compared with the control, the P-glycoproteinexpression levels in the groups transfected with siRNA1,2,3and7reduced 23%（P <0.001）,45%（P <0.001）,36%（P <0.001）,37%（P <0.001）, and29%（P <0.001）, indicating that these siRNA sequences could effectively inhibit theexpression of MDR1gene. The siRNA1was the most effective sequence andit could be used in the subsequent experiment.（3） Further test siRNA1:MTT experiment showed the sensitivity of MCF-7/ADR cells toadriamycin after siRNA1treatment was significantly higher than beforesiRNA1treatment and the difference was significant （P<0.05）. From the lowto high concentrations, the cell sensitivity to adriamycin increased by17%,45%,49%,42%,25%, and1%, respectively. Dose-response relationshipcurve showed that silence efficiency gradually increased with increasedsiRNA1concentrations. The highest inhibition rate was81.7%.（4） Construct siRNA expression vector:SiRNA expression vector was successfully constructed and DNAsequencing showed that the DNA fragment that was inserted into expressionvector had the right sequence.（5） Prepare the siRNA plasmid liposomes and analyze its physiochemicalproperties:Cationic liposomes of siRNA expression plasmid were successfullyprepared. Both the size and electric potential of liposomes could satisfy therequirements and encapsulation rate was as high as88.32%. Electrophoresisshowed that the stability of plasmid liposomes were much greater thanunencapsulated plasmids in DNase-containing solution and in serum,indicating that the entrapped plasmid could be protected effectively byliposomes.（6） Pharmacokinetic study of siRNA plasmid liposomes:Concentration-time curve was set up by depicting the experimental dataas plasmid concentration versus time. After the calculation, the half-life ofplasmid liposomes was about2h and unentrapped plasmid was only about6min.（7） Pharmacodynamic study of siRNA plasmid liposomes in vitro: Realtime PCR showed that no significant difference could be foundbetween the control and negative control. At a dose level of3and6μg,MDR1gene expression levels decreased by33%（P <0.01） and76%（P<0.01）, indicating that siRNA1plasmid liposomes could silence MDR1geneeffectively. The SRB result showed that MCF-7/ADR group and NC grouphad no significant difference. The inhibition of MDR1expression inMCF-7/ADR receiving siRNA1plasmid liposomes group was significantlygreater than in MCF-7/ADR and NC groups with significant differences（P<0.05）. From low to high concentration, the sensitivity of cells toadriamycin increased by10%,6%,14%,23%,44%, and28%.（8） Pharmacodynamic study of siRNA plasmid liposomes in vivo:Drug resistant breast cancer model in SCID nude mice was successfullyestablished. The mouse weight and tumor volume were measured during theexperiment period. Compared with the control group, the body weight of micein other groups decreased, especially in siRNA plasmid liposomes plusadriamycin group, but no statistical difference could be found at each timepoint. No significant difference could be found between adriamycin andcontrol groups. Compared with the control group, the combined treatmentgroup could significantly inhibit tumor growth （P <0.05）. Similar resultscould be achieved by comparing the tumor weight. The tumor inhibition in thecombined therapy group was much greater than adriamycin group. RealtimePCR was carried out to detect the expression levels of MDR1gene in tumortissues. The results showed that compared with the control, adriamycincouldn’t down-regulate the MDR1expression, but the MDR1gene expressionlevels in the combined treatment group reduced by53%（P <0.01）.Conclusions:（1） Four siRNA sequences that could effectively inhibit the expression ofMDR1gene were successfully designed and screened, of which siRNA1hadthe highest interference efficiency.（2） The expression vector of siRNA targeted to MDR1gene（pDual-siRNA1） was successfully constructed, and the cationic liposomes of siRNA expression vector were prepared.（3） SiRNA1and its plasmid liposomes could effectively silence MDR1gene, and the breast cancer cells and tumor growth were significantlyinhibited after treatment with siRNA plasmid liposomes in combination withchemotherapy drug. Multidrug resistance of breast cancer cells was reversedto a great extent.（4） Nanosized cationic liposomes were ideal delivery vehicle of siRNA,which could protect the siRNA from degradation by ribozymes in plasma, andselectively deliver siRNA into the tumor region where it could exert functions.