PDCD4对DAP5表达及大肠癌细胞凋亡的影响

摘要

目的 探讨程序性死亡基因4(PDCD4)对死亡相关蛋白5(DAP5) 的表达及对人大肠癌细胞系LOVO凋亡的影响.方法 构建重组真核表达载体pFLAG/PDCD4,转染人大肠癌细胞LOVO,G418(500 mg/L)筛选获得稳定表达PDCD4的细胞系.RT-PCR及Western blotting检测LOVO细胞中PDCD4的mRNA及蛋白表达,流式细胞仪检测LOVO细胞凋亡情况,Western blotting检测LOVO细胞DAP5表达的变化.结果 成功建立稳定表达PDCD4的大肠癌细胞LOVO-pFLAG/PDCD4.转染PDCD4 的LOVO-pFLAG/PDCD4 组与空白对照LOVO 组、转染空载质粒的LOVO-pFLAG组相比,PDCD4蛋白表达和mRNA水平明显升高(P < 0.01);细胞凋亡率明显增加(P < 0.01);同时伴有DAP5蛋白表达明显升高(P < 0.01).结论 PDCD4能够诱导大肠癌细胞LOVO的凋亡,其机制可能与上调DAP5的表达有关.%Objective To investigate the effect of exogenous programmed cell death 4 (PDCD4) gene on the expression of death associated protein 5 (DAP5) and apoptosis of human colorectal cancer cell LOVO, and involved mechanisms thereof. Methods Recombinant eukaryotic plasmid pFLAG/PDCD4 was constructed and transfected into human colorectal cancer cell LOVO. Cells stably expressing PDCD4 were established by G418 selection (500 mg/L). The levels of PDCD4 protein and mRNA were analyzed by RT-PCR and Western blotting. The apoptotic cells were measured by flow cytometry, and protein expression of DAP5 was detected by Western blotting. Results LOVO-pFLAG/PDCD4 cell line was successfully established by G418 selection. Compared to non-transfection and mock-transfection group, the levels of PDCD4 mRNA and protein were significantly increased, the cell apoptosis ratio was enhanced and the expression of DAP5 protein was increased in transfection group (P < 0.01). Conclusion PDCD4 could induce apoptosis of human colorectal cancer LOVO cells, which mechanism might be involved in up-regulating DAP5 protein.