ABSTRACT
Objective To construct the short hairpin RNA recombinant plasmids targeting mdr1 gene which expresses highly in ovarian cancer resistance strain SKOV3/TAXOL to silence endogenefic mdr1 gene expression and investigate the role of mdr1 gene in the development of resistant ovarian cancer. Methods The pGPU6/GFP/Neo-mdr1 were constructed by gene clone technology. The influence on proliferation and apoptosis were investigated by CCK-8 in SKOV3/TAXOL after transfected pGPU6/GFP/Neo-mdr1. Results The expression against mdr1 proteins were inhibited by pGPU6/GFP/Neo-mdr1. The cell proliferation were inhibited after transfected pGPU6/GFP/Neo-mdr1 by CCK-8. The apoptosis were observed in DAB experiments and the apoptosis rate increased. Conclusion mdr1 plays an important role in proliferation of resistant ovarian cancer and the short hairpin RNA of mdr1 can efficiently suppress mdr1 expression and enhance the apoptosis in SKOV3/ATAXOL.
ABSTRACT
AIM: To explore the feasibility of using vector-based small interfereing RNA(siRNA) to inhibit the expression of MDR1 mRNA and P-glycoprotein and to reverse the multidrug resistance of drug-resistant ovarian cancer cell line.METHODS: An adriamycin-resistant human ovarian cancer cell subline OVCAR/AR was established by stepwise inducement.Another mutidrug-resistant human ovarian cancer cell subline OVCAR/MDR was established by transfecting multidrug resistant gene 1(MDR1) into ovarian carcinoma cell line OVCAR-3.Transfection of MDR1 mRNA specific siRNA expressing plasmids(pSN/mdr1a and pSN/mdr1b) into OVCAR/AR and OVCAR/MDR cells was performed using liposome transfection reagents.MDR1 mRNA expression level was quantified using real time reverse transcription polymerase chain reaction(real time RT-PCR).Flow cytometry(FCM) was performed to assess the expression of p-glycoprotein(P-gp).Multidrug resistant to anticancer agents was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.RESULTS: Basal MDR1 mRNA expression level in drug-resistant cell line OVCAR/MDR was higher than that in OVCAR/AR cell line,and was both higher than that in its parent cell line OVCAR-3.The expression of MDR1 mRNA and P-gp protein in both OVCAR/AR and OVCAR/MDR cells was inhibited dramatically by transfecting with pSN/mdr1a and pSN/mdr1b.The reversal rate of chemoresistance to adriamycin in OVCAR/AR and OVCAR/MDR transfected with pSN/mdr1a and pSN/mdr1b was 79.5% and 93.9%,compared with control group.CONCLUSION: MDR1 expression in the drug-resistant cell lines is partially inhibited by treatment with vector-based MDR1 specific small interference RNAs at the mRNA and protein level,which increases the chemotherapy sensitivity of these drug resistant ovarian carcinoma cell sublines.