水通道蛋白-8对结肠癌细胞凋亡及凋亡抑制蛋白的影响

摘要

目的：通过表达水通道蛋白-8（AQP-8）真核表达载体，观察水通道蛋白AQP-8对HT-29细胞凋亡及凋亡抑制蛋白（ IAPs）表达的影响。方法取对数生长期的人结肠癌HT-29细胞株用于实验。构建AQP-8真核表达载体并转染HT-29细胞，通过Western blot检测GFP-AQP-8转染效率；采用MTT法检测各组细胞增殖抑制率；流式细胞术检测细胞凋亡率；通过Real Time-PCR和Western blot检测转染GFP-AQP-8的HT-29细胞凋亡抑制蛋白（ IAPs）家族成员c-IAP1、c-IAP2、XIAP、NIAP、Survivin和Livin表达水平。结果 Western blot结果显示，GFP-AQP-8转染后结肠癌HT-29细胞AQP-8基因表达显著上调（ P ＜0{．05）。 MTT分析结果显示，转染了GFP-AQP-8的结肠癌HT-29细胞增殖抑制率显著增加（ P ＜0．05）；流式细胞分析发现，转染了GFP-AQP-8的HT-29细胞凋亡率显著增加（ P ＜0．05）。Real Time-PCR和Western blot结果显示，与转染GFP-N1的阴性对照组相比较，转染了GFP-AQP-8的HT-29细胞c-IAP1、c-IAP1、XIAP、Livin和Survivin的mRNA和蛋白表达水平下降（ P ＜0．05），NIAP表达变化不明显（ P ＜0．05）。结论过表达AQP-8可抑制HT-29细胞生长，并能通过下调c-IAP1、c-IAP1、XIAP、Livin和Survivin表达诱导HT-29细胞凋亡。%Objective To investigate the effects of aquaporin-8 ( AQP-8 ) on cell apoptosis and expression of inhibitors of apoptosis proteins( IAPs) by means of eukaryotic expression vector that expresses AQP-8 in human colon cancer HT-29 cells in vitro.Methods Human colon cancer HT-29 cells at logarithm growth period were used in the experiment. AQP-8 eukaryotic expression vector was constructed and transfected into HT-29 cells.Western Blot was used to detect the transfection efficiency;MTT assay was used to detect the cell growth inhibition rate;flow cytometry( FCM) was used to measure the cell apoptosis rate.The expression levels of mRNA and protein of IAPs including c-IAP1,c-IAP2,XIAP,NIAP,Survivin and Livin were detected by fluorescence quantitative Real-time RT-PCR and Western Blot.Results The results by Western Blot showed that the expression levels of AQP-8 were significantly up-regulated in colon cancer HT-29 cells after GFP-AQP-8 transfection ( P <0.05).MTT analysis showed that the proliferation inhibition rate was increased significantly in HT-29 cells after GFP-AQP-8 transfection,as compared with that in GFP-N1 control group ( P <0.05).FCM analysis showed that GFP-AQP-8 transfection obviously induced HT-29 cell apoptosis ( P <0.05 ) .The results of fluorescence Real-time quantitative RT-PCR and Western Blot showed that the expression levels of mRNA and protein of c-IAP1, c-IAP1, XIAP, Livin,and survivin in HT-29 cells were significantly decreased by AQP-8 overexpression ( P <0.05).However,the expression levels of NIAP mRNA and protein had no obvious change ( P >0.05).Conclusion The overexpression of AQP-8 can inhibit growth of HT-29 cell and can induce HT-29 cell apoptosis by down-regulating the expression levels of c-IAP1,c-IAP1,XIAP, Livin and survivin.