人脂肪酸合成酶启动子在乳腺癌细胞中的转录活性分析

Transcriptional activities of human fatty acid synthase promoter in breast cancer cells

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。

AIM： To analyse the transcriptional activities of human fatty acid synthase （FAS） promoter in breast cancer cell lines and normal fibroblast cell line in order to provide some theoretical evidence for the targeted therapy of breast cancer. METHODS： The expression of FAS protein in three breast cancer cell lines （SKBR3, MCF-7 and MDA-MB-231） and normal fibroblast cell line NIH3T3 was examined by Western blot and indirect immunofluorescence assay. The recombinant luciferase reporter vectors pGL3-FAS, pGL3-cerbB2 and pGL3-midkine were constructed and their relative transcriptional activities in SKBR3, MCF-7, MDA-MB-231 and NIH3T3 cell lines were analyzed. RESULTS： FAS protein was expressed in all the three breast cancer cell lines with different levels （SKBR3 〉 MCF-7 〉 MDA-MB-231 ）, but its expression in NIH3T3 was not detected. FAS protein was localized primarily in cytosol. FAS promoter showed relatively high transcriptional activities in breast cancer cell lines which were obviously higher than SV40 promoter and midkine promoter, but it showed little/no in transcriptional activities NIH3T3 cells. The transcriptional activity of FAS promoter was comparable to that of cerbB2 promoter in SKBR3 cells, but it was significantly higher than that in MCF-7 and MDA-MB-231 cell lines. CONCLUSION： FAS promoter can drive therapeutic gene in a wider range of human breast cancers than cerbB2 promoter and it exhibits a stronger activity than midkine promoter. It may serve as a useful tool for transcriptional target of breast cancer gene therapy.