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目的:探讨构建带有肿瘤特异性Survivin启动子的PUMA基因表达载体pUC57-Survivin-PUMA对乳腺癌MCF-7细胞的肿瘤特异性促凋亡作用。方法:化学合成PUMA基因片段克隆至pUC57质粒,构建重组质粒pUC57-PUMA;克隆Survivin启动子序列,取代pUC57-PUMA质粒原有的CMV启动子,构建重组质粒pUC57-Survivin-PUMA,测序鉴定。实验设置pUC57-PUMA组、pUC57-Survivin-PUMA组、阴性对照组(pUC57group)和空白对照组(blank group)。将以上各组分别转染人乳腺癌MCF-7细胞及人正常乳腺Hs 578Bst细胞。转染后48h,western blot技术检测各组细胞中PUMA蛋白表达情况,流式细胞仪检测细胞的凋亡率。结果:重组质粒经测序鉴定证实符合设计要求,构建成功;MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的PUMA蛋白表达均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中只有pUC57-PUMA组的PUMA蛋白表达较其他3组显著增高(P<0.05)。MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的细胞凋亡率分别为29.02%和29.31%,均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中仅pUC57-PUMA组的细胞凋亡率较其他3组显著增高(P<0.05)。结论:Survivin启动子调控的PUMA表达载体能显著增高乳腺癌MCF-7细胞中PUMA的表达,进而促进乳腺癌MCF-7细胞凋亡,而对正常乳腺Hs 578Bst细胞的凋亡不产生影响。
OBJECTIVE: To investigate the tumor-specific pro-apoptotic effects of pUC57-Survivin-PUMA, a PUMA gene expression vector with tumor-specific survivin promoter, on breast cancer MCF-7 cells. METHODS: Chemically synthesized PUMA gene fragment was cloned into pUC57 plasmid to construct recombinant plasmid pUC57-PUMA. The Survivin promoter sequence was cloned to replace the original CMV promoter of pUC57-PUMA plasmid. The recombinant plasmid pUC57-Survivin-PUMA was constructed and sequenced. Experiments were performed with pUC57-PUMA group, pUC57-Survivin-PUMA group, negative control group (pUC57 group) and blank group. The above groups were transfected into human breast cancer MCF-7 cells and human normal breast Hs 578Bst cells, respectively. At 48 hours after transfection, the expression of PUMA protein in each group was detected by western blot, and the apoptosis rate was detected by flow cytometry. RESULTS: The recombinant plasmids were sequenced and confirmed to meet the design requirements and successfully constructed; the PUMA protein expression in pUC57-PUMA group and pUC57-Survivin-PUMA group in MCF-7 cells was significantly higher than that in the control group (P<0.05). Only pUC57-PUMA group had higher PUMA protein expression in breast Hs 578Bst cells than in other three groups (P<0.05). The apoptosis rates of pUC57-PUMA group and pUC57-Survivin-PUMA group in MCF-7 cells were 29.02% and 29.31%, respectively, which were significantly higher than those in the control group (P<0.05), but only in the human normal breast Hs 578Bst cells. The apoptosis rate of pUC57-PUMA group was significantly higher than that of the other three groups (P<0.05). Conclusion: PUMA expression vector regulated by Survivin promoter can significantly increase the expression of PUMA in breast cancer MCF-7 cells, which in turn promotes apoptosis of breast cancer MCF-7 cells, but does not affect the apoptosis of normal breast Hs 578Bst cells.