Reversal Of Multi-drug Resisitance In Paclitaxel Resistant Cell Line SKOV3/TR30 By RNA Interferencing

Objective:To study the efficiency and specifity of MDR1 gene silence in multidurg resistant human ovarian cacinoma cell line(SKOV3/TR30)in vitro by small interference RNA.Methods:1.The MDR1 gene was amplified by PCR,using computer designed MDR1 PCR primer.The amplified fragment was then connected with T vector to construct a plasmid for later DNA sequencing.2.The GAPHD siRNA was transfected into SKOV3/TR30 cells with liposomes, and the GAPDH protein was detected by using Western-blot method,in order to decide siRNA's effective dose and effective time.3.MDR1 specific siRNA was brought into target cells by lipo-transfection.The transcription of MDR1 gene was quantified with FQ-PCR(fluorescence quantitative PCR)and the expression of P-gp was detected with Immunofluroescence technique 48h after transfection.Western-blot was used to detect the P-gp at different time spot after transfection.4.The resistance index and Paclitaxel's IC₅₀was determined by MTT assay.Result:1.SKOV3/TR30 cells were confirmed of highly expression MDR1 gene by PCR.The plasmid was confirmed by restriction enzyme digestion identification and DNA sequencing.MDR1 PCR amplified fragment could be used in later experiments.2.The data of GAPDH siRNA's effective dose and effective time were applied to MDR1 siRNA transfection.MDR1 cDNA PCR result 48h after transfection showed that inhibition was positive.3.FQ-PCR result showed the amount of MDR1 mRNA in silenced cells was about 0.24%of that in not treated ones(p＜0.001).While the GAPDH amount of both cells were almost the same(P＞0.05).No fluorescence could be seen on silenced cells' membrane.The most effective time of silencing was 48h after transfection seen from Western-blot of P-gp.4.Compared to not silenced cells the growth inhibitory rate of silenced ones raised to 46.46%,56.99%from 18.50%,33.68%respectively,at 2noml/L, 1250nmol/L Paclitaxel's concentration(P＜0.05).And Paclitaxel's IC₅₀decreased from 3467.73nmol/L to 344.67nmol/L(P＜0.05).Conlusion:MDR1 mRNA sequence specific siRNA could significantly and specifically inhibit MDR1 mRNA's expression.RNA interference technique is a potent assistant therapy of ovarian cancer chemotherapy.