Expression And Reversal Of Multi-drug Resistance Gene In Ovarian Cancer Cell

ObjectiveIn our study, siRNA targeting to MDR1 was synthesized to investigate the effects of small interference RNA ( siRNA ) on the inhibition of MDR1 mRNA and p-gp expression of ovarian cancer cell of MDR1 gene, and reversal of drug resistance.Methods1.The specific small interfering RNA (siRNA) modified by FAM, was synthrsized 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.The si-GAPDH was used as a positive control of RNAi experiment transfect into SKOV3/AR cell.And detect the decline level of the mRNA and protein of GAPDH.3.Design three siRNA target to MDR1 gene, transfect into SKOV3/AR cell separately and detect the decline level of the mRNA and protein of the target gene in order to screen the most effective siRNA.4.The most effective siRNA was transfected into human ovarian cancer cell line SKOV3/AR by liposome1. The expression of MDR1 mRNA at 48h after transfection was measured by RT-PCR(reverse transcription PCR) and the p-gp expression was detected by flow cytometry 72h after transfection.5.MTT assay was applied to check the drug sensitivity of two different chemotherapeutic agents before and after transfection.Results1.The fluorescence photos show that the best transfection efficiency of SKOV3/AR is 87.80%. 2.48h after siRNA/GAPDH transfected in to SKOV3/AR cells, the GAPDH mRNA was detected by RT-PCR, the results of Blank group,Lopidsome group and Positive control group were 2.46±0.08,2.34±0.04,1.41±0.05, compare to the Blank group, GAPDHmRNA of Positive was significantly decline(p<0.05).3. SiRNA-1 group,SiRNA-2 group,SiRNA-3 group,Blank group,Lopidsome group and Negtive control group MDR1 mRNA expression 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. The outcome of RT-PCR show that each siRNA result in the reduce of expression of target gene respectively(p<0.05), but the inhibitory efficiency is difference. The inhibition ratio of SiRNA-1 group,SiRNA-2 group,SiRNA-3 group to target gene was 43.3%, 41.3% and 58.5%.Acording to our experiment results, siMDR1-3 is screened as the most effective siRNA.4. SiRNA-3 was tranfected into SKOV3/AR cell, p-gp expression of Blank group,Lopidsome group,Negtive control group and SiRNA-3 group was (84.5±0.07)% , (85.35±0.06)% , (84.87±0.01)% , (52.43±0.07)% respectively. Compared with Blank group, the expression level of target gene reduce remarkably, whereas the Negtive control group gene level have no change. Also, the IC50 of SKOV3/AR cell to Adriamycin and Paclitaxel were decline 3.56 and 3 fold respective. ConclusionsRNAi could reversal multi-drug resistance gene in ovarian cancer cell in vitro, it may provided evidence for reversal MDR of solid tumor in vivo.