Mechanism Of ETS2Modulating Transcriptional Activity Of The CXCR4Gene In Breast Cancer Cells

Objective:To investigate whether or how the transcription factor ETS2regulate CXCR4transcription and the molecular mechanism of ETS2modulating transcriptional activity of CXCR4gene in human breast cancer cells.Methods:In MCF-7breast cancer cell lines and MDA-MB-231breast cancer cell lines, through transient transfection, as well as RNAi technology, the expression of ETS2was overexpressed or inhibited. RT-PCR and ELISA was used respectively to detect CXCR4mRNA expression and protein level. Luciferase reporter gene assay was applied to detect CXCR4promoter activity, and ChIP for detecting the amount of ETS2protein binding to CXCR4promoter. Two binding sites of CXCR4promoter were mutated to detect the impact on the activity of CXCR4promoter by gene mutations.Results:After transfected with ETS2expression vector in MCF-7and MDA-MB-231breast cancer cell lines, the mRNA expression and protein level of CXCR4were elevated. The result of luciferase reporter gene assay indicated that ETS2enhanced the expression of CXCR4through activating CXCR4promoter.ChIP assay demonstrated that the amount of ETS2protein binding to CXCR4promoter increased after ETS2transfection. This result indicated that ETS2may activate CXCR4promoter through directly binding with CXCR4promoter. Inhibition of ETS2expression using RNAi could significantly attenuate CXCR4promoter activity, reduce expression of CXCR4and the amount of ETS2binding to CXCR4promoter. Two ETS binding sites of CXCR4promoter were mutated and the result of luciferase reporter gene assay proved that, an arbitrary point mutations attenuated CXCR4promoter activity, while mutation of both binding sites further attenuated CXCR4activity.Conclusion:In MCF-7and MDA-MB-231breast cancer cell lines, overexpression of ETS2could activate CXCR4promoter and the transcription of CXCR4through the combination to the two ETS2binding sites (-540to-535å'Œ-240to-235) of CXCR4promoter. ETS2might be a potential molecular marker of breast cancer metastasis. It is expected to be a new molecular target in the process of diagnosis and treatment of breast cancer.