乳腺癌细胞中人血小板衍生生长因子受体β启动子的基础活性研究

Study of Human PDGFRβ Promoter in Different Human Breast Cancer Cells

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。

In order to determine the basal expression and transcription activites of PDGFRβ promoter in two kinds of breast cancer cells MCF-7 and MDA-MB-231.The basal expression of PDGFRβ in MDA-MB-231 was identified by Western blot and Real-time PCR.Five deletion mutations of human PDGFR-β promoter region were obtained by PCR from human genomic DNA and then cloned into pGL3-basic vector based on Dual-Luciferase reporter system.Two human breast cancer cells MCF-7 and MDA-MB-231 were transfected by the recombinant reporters.Electrophoretic mobility shift assays（EMSA） was used to determine the DNA binding activities of transcription factors.As a result,the region（＋539bp,＋1457bp） showed a positive regulatory role,while the（＋54bp,＋539bp）was negative.The（-983bp,＋54bp）was found to play a significant positive role in MDA-MB-231 whereas there was no effect in MCF-7.The transcription factor AP-1 was observed to highly express in MDA-MB-231 comparing with MCF-7.AP-1 was identified to bind the PDGFRβ promoter region and its binding was higher in MDA-MB-231 than in MCF-7.The fact demonstrate that basal expression and transcription activities of PDGFRβ promoter were different in two types of breast cancer cell and AP-1 played an important role in regulatory of PDGFRβ promoter in breast cancer cells.