乳腺癌中YY1调控FOXM1的生物学功能及分子机制研究

YY1 and FOXM1 can be potentially used as biomarkers of breast cancer diagnosis and prognosis, and therapeutic targets in breast cancer treatment. A previous study indicated that FOXM1 expression was significantly decreased in mouse fibroblast cells with YY1 knockout, suggesting that FOXM1 may be regulated by YY1. However, the underlying molecular mechanism of this regulation has not been explored. The innovative aspect of this project is the investigation of the molecular mechanism underlying YY1-regulated FOXM1 expression, which can provide theoretical insights into developing new approaches of breast cancer therapies. This project has three aims. First, we will determine the effect of altered YY1 expression on FOXM1 levels in breast cancer cells using quantitative real time PCR (qRT-PCR), Western blotting, immunofluorescence, etc. Second, we will explore the mechanisms of YY1-regulated FOXM1 transcription and protein stability using reporter assay, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), ubiquitination assay, co-Iimmunoprecipitation (Co-IP), etc. Third, with ectopic expression and gene silencing of YY1 and/or FOXM1, we will evaluate the effects of YY1/FOXM1 signal axis on proliferation, migration, and invasion of breast cancer cells in vitro and tumor formation in a xenograft mouse model. This study will delineate the biological function and molecular mechanisms of YY1-regulated FOXM1 expression in breast cancer. Importantly, the achievement of this project will likely reveal novel therapeutic targets for clinical treatment of breast cancer patients.

YY1和FOXM1可作为乳腺癌临床诊断和预后的生物标志物，以及治疗的潜在靶标。有文献报道，小鼠模型中YY1敲除伴随着FOXM1表达下降，提示乳腺癌中FOXM1可能受到YY1调节，然而，其调节机制还未被研究。本项目的创新点在于，剖析YY1调节FOXM1表达的分子机制，为建立治疗乳腺癌新方法提供理论依据。研究内容包括：第一，利用定量PCR、蛋白免疫印记和免疫荧光等手段验证乳腺癌细胞中改变YY1对FOXM1表达的影响；第二，利用报告载体、凝胶迁移实验、染色质免疫共沉淀、泛素化实验、免疫共沉淀等方法探究YY1是否在转录和蛋白水平调节FOXM1及其调控机制；第三，利用乳腺癌细胞和移植瘤裸鼠模型，通过外源表达、RNA干扰等技术检测YY1/FOXM1信号轴对乳腺癌细胞增殖、迁移、侵袭以及移植瘤生长等的影响。以上研究将揭示乳腺癌中YY1调控FOXM1的生物学功能及分子机制，为乳腺癌临床治疗提供新的靶点。

YY1和FOXM1可作为乳腺癌临床诊断和预后的生物标志物，以及治疗的潜在靶标。有文献报道，小鼠模型中YY1敲除伴随着FOXM1表达下降，提示乳腺癌中FOXM1可能受到YY1调节，然而，其调节机制还未被研究。本项目的研究内容和实验结果包括：1、利用定量PCR、蛋白免疫印记和免疫荧光等手段验证乳腺癌细胞中改变YY1对FOXM1表达的影响。结果发现，外源表达YY1可显著促进MCF-10A细胞中FOXM1的表达。然而，敲低YY1表达，可明显降低乳腺癌细胞MDA-MB-231和MCF-7中FOXM1的表达。乳腺癌中FOXM1可作为YY1的一个关键下游靶基因。2、第二，利用报告载体、凝胶迁移实验、染色质免疫共沉淀、泛素化实验、免疫共沉淀等方法探究YY1是否在转录和蛋白水平调节FOXM1及其调控机制。结果发现，乳腺癌细胞中YY1可直接与FOXM1启动子区结合，促进其转录表达。同时，YY1可直接与FOXM1蛋白结合，阻碍E3连接酶适配体CDH1介导的FOXM1泛素化-蛋白酶体降解，从而促进FOXM1蛋白稳定。乳腺癌中YY1可从转录和翻译后水平促进FOXM1的表达。第三，利用乳腺癌细胞和移植瘤裸鼠模型，通过外源表达、RNA干扰等技术检测YY1/FOXM1信号轴对乳腺癌细胞增殖、迁移、侵袭以及移植瘤生长等的影响。结果发现，YY1通过上调FOXM1的表达，促进乳腺癌细胞的增殖、克隆形成以及迁移等生物学功能。此外，在本项目延申的研究中，我们发现，1）YY1通过与转录共激活因子形成相分离复合体，促进超级增强子形成，进而激活下游基因表达。2）YY1与FOXM1蛋白结合主要依赖FOXM1氨基端结构域的D box和KEN box区域。3）FOXM1蛋白发生泛素化的位点是K48和K63。以上结果，解析了乳腺癌中YY1调控FOXM1表达的生物学功能及分子机制，为后续靶向YY1和FOXM1的药物研发提供理论基础。