hTERT启动子的克隆及在MCF7乳腺癌细胞中的特异转录活性

Cloning of hTERT promoter and its specific transcriptional activity in MCF7 breast cancer cell

背景与目的利用肿瘤特异性启动子增强肿瘤细胞中治疗基因的转录选择性是达到基因治疗靶向性的有效途径。人端粒酶催化亚基(hTERT)在大部分肿瘤细胞中呈特异性高表达[1-2],有可能成为肿瘤特异性启动子。本文扩增了hTERT基因启动子并分别克隆于报告基因重组质粒pEGFP-1和pGL3-Basic,检测并证实hTERT启动子在乳腺癌细胞MCF7中的特异转录活性。方法采用套式PCR法扩增hTERT启动子,将其分别克隆到含报告基因增强绿色荧光蛋白(EGFP)的重组质粒pEGFP-1和含Luciferase的重组质粒pGL3-Basic,经脂质体分别转染人乳腺正常上皮细胞系HBL100和乳腺癌细胞MCF7,荧光显微镜观察EGFP的表达情况,并测定两种细胞中荧光素酶转录表达差异。结果与正常细胞HBL100相比,hTERT启动子在MCF7细胞呈现强的绿色荧光表达;荧光素酶活性检测与其一致,hTERT启动子在MCF7乳腺癌细胞的荧光素酶相对活性RLU/U值(33784)约相当于HBL100细胞中(2400)的15倍。结论hTERT启动子在乳腺癌MCF7细胞中具有很强的特异性,为进一步开发肿瘤的特异性基因治疗奠定实验基础。

Background and purpose： Gene therapy is a novel approach for the treatment of the patients with breast cancer. One of the effective ways is to direct transgenic expression to specific tissues or tumors with the use of tissue-specific-promoters （TSP）. hTERT （ human telomerase reverse transcriptase） is highly expressed in many types of cancers including breast cancer. Thus, we hypothesized that the hTERT promoter targeting with gene therapy vectors could be exploited for breast cancer, in this study, we amplified hTERT gene promoter and cloned it into the reporter vector pEGFP and pGL3-Basic. Afterwards, the specific transcription of hTERT promoter in MCF7 cells was evaluated. Methods： hTERT gene minimal promoter was PCR amplified and cloned into the reporter plasmid pEGFP-1 and pGL3-Basic. The constructs pEGFP/TERT and pGL3/TERT were transfected into MCF7 breast cancer cells and HBL100 human epithelial cells, respectively. The expression of EGFP and luciferase were investigated, respectively.. Results： pEGFP/TERT and pGL3 / TERT bearing hTERT gene promoter were constructed. The specific expression of EGFP was detected in MCF7 cells while little expression of EGFP was seen in HBL100 cells. In accordance with EGFP, luciferase driven by hTERT also showed specific and high activity in MCF7 cell （ RLU/U： 33784）, which is 15 times higher than in HBL100 （ RLU/U： 2400）. Conclusions： The high transcriptional activity of hTERT gene promoter in MCF7 cell indicates its potential utility as a novel candidate for transcriptional targeting of breast cancer.