下调X连锁调亡抑制蛋白基因表达增强多西他赛诱导膀胱癌细胞凋亡的作用

Effects of down-regulation of X-linked inhibitor of apoptosis protein gene on docetaxel-induced apoptosis in bladder cancer cells

目的:观察人源性膀胱癌细胞在下调X连锁凋亡抑制蛋白(XIAP)基因表达后,多西他赛对其敏感性的影响。方法:以shRNA和shRNA-XIAP质粒分别稳定转染人源性膀胱癌T24T细胞。以荧光显微镜观察转染细胞。以RT-PCR和Western blotting法分别检测膀胱癌细胞XIAPmRNA和蛋白的表达。与T24T以及转染空载体的T24T细胞相比较,以ATPase法检测多西他赛对转染XIAP基因的膀胱癌细胞的细胞毒性。相差显微镜下观察,流式细胞术检测多西他赛预处理转染XIAP基因的膀胱癌细胞的凋亡率;以Western blotting法检测细胞内的聚腺苷二磷酸核糖聚合酶(PARP)和caspase-3蛋白表达和裂解水平。结果:荧光显微镜下分别观察shRNA和shRNA-XIAP转染的T24T细胞,均见稳定荧光。Western blotting和RT-PCR检测结果显示,人源性膀胱癌T24T细胞在稳定转染反义XIAP基因后,其XIAP蛋白和mRNA水平均显著降低。经多西他赛处理24 h后,转染反义XIAP基因的T24T细胞IC_(50)为(1.23±0.62)nmol/L,远低于T24T以及转染空白载体的对照组细胞[(8.22±1.23)nmol/L,(8.35±0.98)nmol/L,P<0.01]。流式细胞术检测结果显示,T24T shRNA-XIAP细胞组(2 nmol/L和5nmol/L)凋亡率[(41.45±6.23)%和(74.82±5.46)%]显著高于转染空载体细胞的凋亡率[(25.34±3.81)%和(34.14±6.25)%,P<0.01]。与转染空载体的细胞相比较,T24T shRNA-XIAP细胞内的PARP和caspase-3明显降低。结论:下调膀胱癌细胞的XIAP基因表达后,可显著增强化疗药物多西他赛所诱导的膀胱癌细胞的凋亡,并增强多西化赛的细胞毒性。

AIM：To observe the effects of down-regulation of X-linked inhibitor of apoptosis protein（XIAP） gene on the chemotherapeutic sensitivity of bladder cancer cells.METHODS：The shRNA and shRNA-XIAP were transfected into bladder cancer T24T cells.The fluorescence microscopy was used to detect the transfection effects.XIAP gene expression was detected by RT-PCR and Western blotting.Docetaxel was administrated to T24T,T24T shRNA and T24T shRNA-XIAP bladder cancer cells.ATPase methods was performed to measure in vitro cell viability.Morphological changes and apoptotic rates were determined by phase contrast microscopy and flow cytometry assay.Cellular poly（ADPribose） polymerase（PARP） and caspase-3 protein expression and their cleavage were assayed by Western blotting.RESULTS： The fluorescence was obvious in the T24T shRNA and T24T shRNA-XIAP cells under the fluorescence microscope. Using Western blotting and RT-PCR methods,the protein and mRNA levels of XIAP gene in T24T shRNA-XIAP cells were significantly decreased,respectively.After treated with various concentrations of docetaxel for 24 h,the IC_（50） of T24T shRNA-XIAP cells was（1.23±0.62） nmol/L,lower than that of T24T and T24T shRNA cells,which were （8.22±1.23 ） and（8.35±0.98 ） nmol/L（P〈0.01）,respectively.Compared with control group,the typical morphological changes of apoptosis were observed in T24T shRNA-XIAP cells.Detected by flow cytometry assay,the apoptotic rates in shRNA -XIAP group were（41.45±6.23）%and（74.82±5.46）%after exposed to docetaxel at the concentrations of 2 nmol/L and 5 nmol/L for 24 h,which were significantly higher than that in T24T shRNA group with（25.34±3.81）%and（34.14±6.25）%,respectively（P〈0.01）.Compared with T24T shRNA cells,the cleavage of PARP and caspase-3 proteins in the cells transfected with shRNA-XIAP was significantly increased.CONCLUSION：XIAP gene is significantly down-regulated via shRNA-XIAP,which could increase the docetaxel-induced apoptosis and cytotoxic activity.