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目的分析人脂肪酸合成酶(FAS)启动子在乳腺癌细胞中的转录活性,并与cerbB2启动子、midkine启动子相比较,为其乳腺癌靶向基因治疗提供依据。方法Western blot和间接免疫荧光法检测FAS蛋白在3种人乳腺癌细胞系SKBR3、MCF-7、MDA-MB-231以及正常成纤维细胞系NIH3T3中的表达。构建pGL3-FAS、pGL3-cerbB2和pGL3-midkine荧光素酶表达载体,并分析其在SKBR3、MCF-7、MDA-MB-231和NIH3T3细胞系中的相对转录活性。结果FAS蛋白在3种乳腺癌细胞系中均有表达,其中在SKBR3细胞中表达量最高,在MDA-MB-231细胞中表达量最低,而在正常成纤维细胞系NIH3T3中不表达。FAS蛋白主要定位于细胞质。FAS启动子在3种乳腺癌细胞系中均具有较强的转录活性,转录活性明显高于强启动子SV40和肿瘤特异性启动子midkine;而在正常成纤维细胞中,转录活性很低。FAS启动子在cerbB2高表达的SKBR3细胞中转录活性最高,与cerbB2启动子相当;在cerbB2表达较弱的MCF-7和MDA-MB-231细胞中的转录活性则明显高于cerbB2启动子。结论FAS启动子在乳腺癌细胞中具有强转录活性,较cerbB2启动子作用范围更广,较midkine启动子转录活性更高,而在正常细胞中转录活性很低,具有良好的乳腺癌靶向性,可作为乳腺癌广谱基因治疗的靶向工具。
Objective To analyze the transcriptional activity of human fatty acid synthase (FAS) promoter in breast cancer cells, and to provide a basis for targeted breast cancer gene therapy compared with cerbB2 promoter and midkine promoter. Methods The expression of FAS protein was detected by Western blot and indirect immunofluorescence in three human breast cancer cell lines, SKBR3, MCF-7, MDA-MB-231 and normal fibroblast cell line NIH3T3. The pGL3-FAS, pGL3-cerbB2 and pGL3-midkine luciferase expression vectors were constructed and analyzed for their relative transcriptional activity in SKBR3, MCF-7, MDA-MB-231 and NIH3T3 cell lines. Results FAS protein was expressed in all three breast cancer cell lines. Among them, the expression level of FAS protein was the highest in SKBR3 cells, the lowest in MDA-MB-231 cells, but not in the normal fibroblast cell line NIH3T3. FAS protein mainly located in the cytoplasm. The FAS promoter has strong transcriptional activity in all three breast cancer cell lines, and the transcriptional activity is significantly higher than that of the strong promoter SV40 and the tumor specific promoter midkine. In normal fibroblasts, the transcriptional activity is low. The transcriptional activity of FAS promoter in SKBR3 cells with high cerbB2 expression was comparable with that of cerbB2 promoter. The transcriptional activity of FAS promoter in MCF-7 and MDA-MB-231 cells with weak cerbB2 expression was significantly higher than that of cerbB2 promoter. Conclusion The FAS promoter has strong transcriptional activity in breast cancer cells, which has a broader range of action than cerbB2 promoter, higher transcriptional activity than midkine promoter, low transcriptional activity in normal cells and good breast cancer targeting , Can be used as a broad-spectrum breast cancer gene therapy tools.