抑制c-FLIP_L表达增强rhTRAIL蛋白杀伤肺癌A549细胞的研究

目的:探讨抑制c-FLIPL表达增强rhTRAIL蛋白杀伤肺癌A549细胞株的可行性。方法:构建c-FLIPL序列特异性siRNAs表达载体并转染A549细胞抑制c-FLIPL基因表达,化学抑制剂他莫昔芬抑制c-FLIPL蛋白表达。RT-PCR和Western blot法检测c-FLIPL的mRNA和蛋白表达的变化。Western blot法检测caspase-8的活化情况。流式细胞仪检测A549细胞凋亡率,MTT法检测联合应用RNAi+rhTRAIL和他莫昔芬+rhTRAIL对A549细胞的杀伤活性。结果:特异性siRNA片段能显著降低A549细胞中c-FLIPL的mRNA水平和蛋白水平。他莫昔芬可以显著抑制c-FLIPL蛋白的表达。两者均可以实现caspase-8的活化,并促进rhTRAIL对A549细胞诱导凋亡作用,凋亡率从2.52%提高到64.5%和59.2%(P<0.05),他莫昔芬和siRNA均可显著促进rhTRAIL蛋白对A549细胞的增殖抑制作用,抑制率分别提高到73.2%和69.5%(P<0.05)。结论:靶向抑制FLIP蛋白表达能增加A549细胞对rhTRAIL诱导细胞凋亡的敏感性,有利于促进rhTRAIL蛋白在治疗NSCLC方面的研究应用。

Inhibition of c-FLIP_L enhance rhTRAIL-based therapies for A549 cells

Objective:To investigate the possibility of enhanced killing activity of rhTRAIL to A549 cells by inhibition of c-FLIPL activity.Methods:siRNAs specific for c-FLIPL sequence and chemical inhibitor tamoxifen were added to inhibit the expression and activity of c-FLIPL and then the application of rhTRAIL protein further killed A549 cells.Caspase-8 was detected by Western blot method.The apoptotic rate of A549 cells was examined by FACS.Cell cytotoxicity activity was detected by MTT assay.Results:Short siRNAs targeting FLIP down-regulated the mRNA and the protein level of c-FLIPL.Tamoxifen inhibited the protein level of c-FLIPL.c-FLIPL-siRNA and tamoxifen may all induce the activity of caspase-8 and promote apoptosis rate of A549 cells to 64.5% and 59.2%.A significant reduction of the rate of cell proliferation was observed and inhibition rate was 73.2% and 69.5%(P<0.05).Conclusion:Inhibition of c-FLIPL protein increases the sensitivity of the A549 cell to rhTRAIL and is conducive to the promotion of rhTRAIL protein in the treatment of NSCLC applications.