论文部分内容阅读
目的:探讨低迁移表型的乳腺癌细胞MCF-7和高迁移表型的乳腺癌细胞MDA-MB-231中血小板衍生生长因子β启动子的基础活性及转录调控差异。方法:Real-Time PCR,Weastern blot等技术检测PDGFRβ在2株细胞中的转录和表达差异。双荧光报告系统检测PDGFRβ启动子各缺失突变片段在2株细胞中的活性,筛选差异片段。生物信息学预测启动子区可能存在的转录因子。凝胶迁移实验研究转录因子在两株乳腺癌细胞中对PDGFRβ启动子的调节活性。结果:两株细胞中都有PDGFRβ的内源性表达,且在MDA-MB-231中表达较高。在2株细胞中找到了人PDGFRB启动子的重要活性调节区,(+539bp,+1457bp)在2株细胞中均呈负调控,(+54bp,+539bp)在两株细胞中均呈正调控,(-983bp,+54bp)在MDA-MB-231中呈显著正调控,在MCF-7中没有活性。转录因子AP1的转录活性和与DNA的结合活性在MDA-MB-231中均高于MCF-7。结论:不同迁移表型的乳腺癌细胞中PDGFRβ存在不同的表达调控机制,PDGFRβ启动子活性片段(-983bp,+54bp)在两株细胞中存在显著活性差异。转录因子AP-1在两株细胞中有表达水平和结合活性差异。
OBJECTIVE: To investigate the basal activity and transcriptional regulation of platelet-derived growth factor β promoter in breast cancer cells MCF-7 with low migration phenotype and MDA-MB-231 breast cancer with high migration phenotype. Methods: Real-Time PCR and Weastern blot were used to detect the transcription and expression of PDGFRβ in two cell lines. Dual fluorescence reporter system detects PDGFRβ promoter deletion mutant in two cell activity, screening for different fragments. Bioinformatics predicts possible transcription factors in the promoter region. Gel Migration Assay The regulatory activity of the transcription factor on the PDGFR [beta] promoter in two breast cancer cells was investigated. RESULTS: Both cells had endogenous expression of PDGFRβ and higher expression in MDA-MB-231. The major active regulatory region of human PDGFRB promoter was found in 2 cells (+ 539 bp, + 1457 bp) and negatively regulated in both cells (+ 54bp, + 539 bp) in both cell lines, (-983bp, + 54bp) showed a significant positive control in MDA-MB-231 and no activity in MCF-7. Transcription factor AP1 transcriptional activity and DNA binding activity in MDA-MB-231 were higher than MCF-7. CONCLUSION: PDGFRβmRNA expression differs in breast cancer cells with different migration phenotypes. The active fragment of PDGFRβ promoter (-983bp, + 54bp) has significant difference in the two cell lines. The transcription factor AP-1 has difference in expression level and binding activity in both cells.