RNAi靶向沉默FLIP基因促进TRAIL诱导卵巢癌A2780细胞凋亡

Promotion of TRAIL-induced apoptosis by siRNA to FLIP gene in ovarian cancer cell line A2780

目的探讨RNAi靶向沉默FLIP基因对TRAIL诱导卵巢癌细胞凋亡的影响。方法设计并体外化学合成FLIP序列特异性双链RNA,在脂质体(LipofectamineTM2000)介导下转染人卵巢癌细胞株A2780。采用半定量RT-PCR和Western blot法检测FLIP-siRNAs转染前后A2780细胞FLIPmRNA和蛋白表达的变化,并筛选出抑制作用最强的FLIP-siRNA。四甲基偶氮唑盐(MTT)法检测FLIP-siRNA转染前后TRAIL对A2780细胞生长抑制作用的变化。以Annexin-V-PI双染法流式细胞术(FCM)比较FLIP-siRNA转染前后TRAIL诱导的细胞凋亡的情况。结果特异性FLIP-siRNA片段能有效降低A2780细胞中FLIPmRNA和蛋白水平(P<0.01),并具有时间依赖性;转染FLIP-siRNA后,TRAIL对A2780细胞的生长抑制作用明显增强(P<0.05),TRAIL诱导的细胞凋亡也显著增加(P<0.05)。结论靶向FLIP基因的siRNAs可在转录和翻译水平抑制FLIP表达,并可增加细胞对TRAIL的敏感性。

Objective To investigate the effect of FLIP-siRNA-mediated gene silencing on the sensitivity of ovarian cancer cells to TRAIL. Methods Designed and synthesized three siRNAs based on the sequence of FLIP mRNA. Then transfected them into ovarian cancer cell line A2780 with LipofectamineTM 2000. FLIP mRNA and protein 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 induced by TRAIL were examined by MTF assay and flow cytometry. Results Short siRNAs targeting FLIP down-regulated mRNA and the protein level of FLIP oncogene in a time-dependent manner（ P 〈 0. 01 ）. Cell growth inhibition and apoptosis efficiency induced by TRAIL were enhanced by FLIP-siRNA transfection （P 〈 0. 05 ）. Conclusion FLIP-siRNAs can effectively inhibit FLIP expression at transcriptional and translational level and greatly enhance TRAIL sensitivity of ovarian cancer cells.