Sensitizing Ovarian Cancer Cell For TRAIL-Induced Apoptosis By Small Interfering RNAs Of FLIP Gene

Objective:To investigate the effect of FLIP-siRNA-mediated gene silencing on bionomics of ovarian cancer cell line A2780 and the sensitivity of A2780 cells to TRAIL.To approach the role of FLIP in TRAIL-induced apoptotic pathway.Our studies may provide a new insight for the treatment of ovarian cancers.Methods:Design and chemically synthesize three siRNAs based on the sequence of FLIP mRNA.They were transfected into ovarian cancer cell line A2780 with Lipofectamine^TM2000.The FLIP mRNA and protein level were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.The FLIP-siRNA which has the most powerful inhibition was selected.Cell growth inhibition and apoptosis were observed with MTT assay and flow cytometry, respectively.After transfection for 48h,cells were treated with Recombinant human soluble TRAIL at a concentration of 50,100,250 nmol/ml for another 24h..Cell growth inhibition and apoptosis induced by TRAIL were observed with MTT assay and flow cytometry.Results:1.FLIP_L-siRNAs transfection and FLIP_L mRNA expression in A2780 cells.FLIP_L-siRNAs were transfected into ovarian cancer cell line A2780 with Lipofectamine^TM2000.After the FLIP_L-siRNAs were transfected for 48h,FLIP_L mRNA expression was detected by RT-PCR.The result showed that the expression of FLIP_L mRNA was reduced after FLIP_L-siRNAs were transfected.The inhibition rates of FLIP_L-1,FLIP_L-2 and FLIP_L-3 were(77.4±1.2)%,(67.1±1.1)%and(48.3±0.9)%,which were obviously higher than that of the control groups(P＜0.05).Among the three FLIP_L-siRNAs,the FLIP_L-1 is most effective(P＜0.05).2.FLIP_L protein expression in A2780 cells.After the A2780 cells transfected for 48h, FLIP_L protein expression in A2780 cells weie detected by Western blot.The result showed that the expression of FLIP_L protein was reduced after FLIP_L-siRNAs were transfected.The inhibition rates of FLIP_L-1,FLIP_L-2 and FLIP_L-3 were(66.1±1.3) %,(48.6±1.5)%and(36.2±1.6)%,which were obviously higher than that of the control groups(P＜0.05).Among the three FLIP_L-siRNAs,the FLIP_L-1 is most effective(P＜0.05).3.Cell growth inhibition by FLIP_L-siRNA transfection.Cell growth inhibition rate of A2780 cells was detected by MTT colorimetry after FLIP_L-1 transfection for 24,36,48h.The result showed that the cell growth inhibition rates of A2780 cells after FLIP_L-1 transfection for 24,36,48h were(7.8±0.3)%,(9.5±0.8)%and(11.2±0.9) %,which were obviously higher than that of the control groups(P＜0.05).4.The effect of FLIP_L-siRNA transfection on apoptosis of A2780 cells.Apoptotic rate of A2780 cells was detected by Annexin-V-PI Flow Cytometry(FCM)after FLIP_L-1 transfection for 24,36,48h.The result showed that the apoptotic rates of A2780 cells after FLIP_L-1 transfection for 24,36,48h were(4.63±0.49)%,(6.14±0.55)% and(7.39±0.73)%,which were obviously higher than that of the control groups(P＜0.05).5.Cell growth inhibition by TRAIL after FLIP_L-siRNA transfection.After the FLIP_L-1 were transfected for 48h,cells were treated with Recombinant human soluble TRAIL at a concentration of 50,100,250μg/L and then cultured for additional 24h until they were ready to assay.Cell growth inhibition by TRAIL was detected by MTT colorimetry.The result showed that the cell growth inhibition rates of A2780 cells which were treated by FLIP_L-1 and TRAIL(50,100,250μg/L)were (11.9±0.28)%,(16.6±0.40)%and(21.5±0.74)%,with significant differences compared with control groups(P＜0.05).6.The effect of FLIP_L-siRNA transfection on TRAIL-induced A2780 cells apoptosis. After the FLIP_L-1 were transfected for 48h,cells were treated with Recombinant human soluble TRAIL at a concentration of 50,100,250μg/L for additional 24h. Apoptosis was detected by Annexin-V-PI Flow Cytometry(FCM).The result showed that the apoptotic rates of A2780 cells which were treated by FLIP_L-1 and TRAIL(50,100,250μg/L)were(8.72±0.92)%,(11.40±0.96)%and(16.51±0.69)%, with significant differences compared with control groups(P＜0.05).Conclusion:1.FLIP-siRNA can significantly down-regulate the FLIP expression in transcriptional and translational level.2.RNAi-mediated FLIP gene silencing can inhibit cell growth and induce cell apoptosis in ovarian cancer cell line A2780.3.RNAi-mediated FLIP gene silencing can greatly enhance TRAIL sensitivity in ovarian cancer cell line A2780.