Survivin基因启动子的克隆及在乳腺癌细胞MCF7中的特异转录活性研究

CLONING OF SURVIVIN PROMOTER AND ITS SPECIFIC TRANSCRIPTIONAL ACTIVITY IN MCF7 BREAST CANCER CELLS

目的研究Survivin基因启动子在乳腺癌细胞MCF7中的特异转录活性。方法采用非限制酶方法获得具有酶切黏性末端的Survivin最小启动子,将其克隆到含有报告基因增强绿色荧光蛋白(enhanced greenfluorescent protein,EGFP)的载体pEGFP-1和含有荧光素酶Luciferase的质粒载体pGL3-Basic,经脂质体分别转染人正常乳腺上皮细胞系HBL-100和乳腺癌细胞MCF-7,荧光显微镜下观察EGFP的表达情况,同时测定2种细胞中荧光素酶表达的差异。结果 Smvivin启动子在MCF7细胞中呈现高转录活性;而在HBL100细胞中转录活性极低。结论 Survivin启动子在肿瘤细胞MCF7中具有很强的特异性,为进一步开发肿瘤特异性基因治疗奠定了实验基础。

Objective To study the specific transcriptional activity of Survivin promoter in MCF - 7 breast cancer cells. Methods A 260 - bp fragment harboring Survivin gene minimal promoter was amplified in two independent PCR amplifications, in which the PCR product was obtained with specific blunt end restriction terminus in either upstream or downstream of the PCR product. The two PCR products were directly annealed to generate the insert DNA with adhesive restriction terminus at both ends, which was subsequently cloned into the reporter plasmid pEGFP - 1 and transfected into MCF -7 cells and HBL- 100 cells respectively. The expressions of enhanced green fluorescent protein （EGFP） and luciferase were investigated. Results At 20h post transfection, the specific expression of EGFP was detected in MCF7 cells while little expression of EGFP was in HBL100 cells. In accordance with EGFP, luciferase driven by Survivin promoter also showed specific and high activity. Conclusion The high transcriptional activity of Survivin gene promoter in MCF7 cells indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.