The In Vivo Study Of Small Interfering RNA Targeting MDR1 Treating Ovarian Cancer

ObjectiveTo further validate a knockdown approach for circumventing the multidrug resistance gene and the influence on tumor growth,we used small interfering RNA targeting MDR1 gene to inhibit the expression of MDR1 gene and P-glycoprotein in vivo.Methods1. The specific small interfering RNA modified by FAM,was synthesized and transfected into SKOV3/AR cells by lipofectmine 2000.The transfection efficiency of siRNA was evaluated by calculating the ratio of green fluorescent to total cell using image analysis software,and got the optimal transfection concentration of siRNA.2. Three small interfering RNAs targeting MDR1 were designed and then transfected into human ovarian cancer cell line SKOV3/AR by lipofectamine respectively.After 48 h,the transfected cells were harvested for RT-PCR to select the most effective siRNA.3. Ascite tumor xenografts were established by implanting human ovarian carcinoma cells SKOV3/AR intraperitoneally into the nude mice.When the tumor models were found with ascites,the mice were randomized into the following three treatment groups with each group six mice respectively:Taxol,Taxol with lipofectamine and Taxol with siRNA/MDR1- lipofectamine intraperitoneal injection. During the course of treatment,the mice were weighed twice a week to evaluate the toxicity of the treatment. 4. The tumor growth rate and the ascite growth rate of mice were investigated.The expressions of MDR1 gene and P-gp in mice were determined by reverse transcription-polymerase chain reaction and immunohistochemistry respctively.Results1. The fluorescence photos show that the transfection efficiency was the best when the concentration of siRNA was 30nM.2. MDR1-mRNA expressions in cells transfected after 48 h:the MDR1-mRNA expression of siRNA/MDR1-I group,siRNA/MDR1-II group,siRNA/MDR1-III group,Blank group,Lipofectamine group and Negtive control group was 1.98±0.03,2.05±0.09,1.45±0.08,3.49±0.28,3.46±0.17 and 3.48±0.26 respectively;compared to the control group,the MDR1-mRNA expression of each siRNA group was significantly decreased(p<0.05),and we found that the MDR1-mRNA expression in siRNA/MDR1-III group was most effectively inhibited,so we utilized siRNA/MDR1-III for the later experiment.3. After 20 days,the mice were found with ascites.During the treatment,there was no significant toxicity with the siRNA treatment.The average tumor weight and ascite volume decreased by 43.6% and 29.7% in the group treated with Taxol and siRNA/MDR1-lipofectamine compared with those treated with Taxol alone (P<0.001).The expressions of MDR1 gene and P-gp in the group treated with Taxol and siRNA/MDR1-lipofectamine were also decreased compared with those in the group treated with Taxol alone (P<0.001).ConclusionsSmall interfering RNA targeting-MDR1 can effectively and specifically suppress the expression of MDR1(P-glycoprotein) and inhibit ovarian cancer growth in vivo; siRNA technology may become a potent and safe gene therapy for human ovarian carcinoma in future.