FOXA1介导EAF2调控雄激素受体活性以及前列腺癌细胞增殖、迁移的分子机制研究
发布时间：2019-02-16 09:21
【摘要】：目的:ELL-相关因子2(EAF2)是前列腺癌中重要的肿瘤抑制因子,然而其抑癌作用的具体机制尚不清楚。蛋白间的相互作用是调控其功能的重要方式之一。本研究拟通过筛选并鉴定前列腺癌中与EAF2相互作用的蛋白,探讨EAF2调控雄激素受体活性及前列腺癌细胞增殖、迁移的分子机制。方法:应用线虫(C.elegans)模型进行RNAi筛查,寻找与eaf-1发挥协同作用的基因。应用免疫共沉淀实验验证EAF2与筛选蛋白的结合情况。通过realtime PCR和剂量依赖共表达实验研究EAF2对所选蛋白表达的调控作用及机制。利用si RNA基因干扰技术对前列腺癌细胞中EAF2和所选蛋白进行单独敲除或双敲除,realtime PCR和双荧光素酶报告基因检测AR下游基因的表达水平以明确AR转录活性变化;染色质免疫共沉淀实验检测EAF2和筛选蛋白相互作用对AR与下游基因DNA结合能力的影响;Brd U、克隆形成实验和transwell细胞迁移实验检测细胞增殖和迁移能力的改变。结果:以线虫生育能力作为参考指标,我们筛选出5个与eaf-1(人类EAF2基因的C.elegans同源基因)具有协同作用的基因,分别为lin-53,pha-4,hmg-1.2,ruvb-1和set-6,其中pha-4的人类同源基因FOXA1在前列腺癌进展中具有重要作用,因此我们选择其作为后续研究基因。在前列腺癌细胞中,EAF2与FOXA1相互结合。敲除前列腺癌细胞LNCa P中的EAF2能够上调内源性FOXA1的蛋白水平,过表达EAF2则能降低FOXA1的表达。EAF2对FOXA1的m RNA表达无影响。敲除LNCa P细胞的EAF2表达能够上调AR下游基因表达水平,促进AR与ARE结合,并增强细胞的增殖和迁移能力;对FOXA1和EAF2进行双敲除则能够抵消单独敲除EAF2对AR下游基因表达、AR同ARE的结合、LNCa P细胞增殖和迁移能力的促进作用。表明FOXA1在EAF2对AR转录活性、细胞增殖和迁移的调控中起介导作用。结论:在EAF2调控前列腺癌细胞AR信号通路、细胞增殖和迁移能力的过程中,FOXA1发挥了重要的介导作用。
[Abstract]:Objective: ELL- related factor 2 (EAF2) is an important tumor suppressor in prostate cancer. The interaction between proteins is one of the important ways to regulate its function. The aim of this study was to study the molecular mechanism of EAF2 regulating androgen receptor activity and proliferation and migration of prostate cancer cells by screening and identifying proteins interacting with EAF2 in prostate cancer. Methods: the C.elegans model was used to screen RNAi and search for genes that could play a synergistic role with eaf-1. The binding of EAF2 to screened proteins was verified by immunoprecipitation assay. The regulation and mechanism of EAF2 on the expression of selected proteins were studied by realtime PCR and dose-dependent co-expression experiments. Si RNA gene interference technique was used to detect the expression level of AR downstream gene in prostate cancer cells by single knockout or double knockout, realtime PCR and double luciferase reporter gene in order to clarify the transcriptional activity of AR. Effect of EAF2 and screening protein interaction on the binding ability of AR to downstream gene DNA by chromatin immunoprecipitation assay; Brd U, clone formation assay and transwell cell migration assay were used to detect the changes of cell proliferation and migration ability. Results: using nematode fertility as a reference index, we screened out five genes that have synergistic effect with eaf-1 (C.elegans homologous gene of human EAF2 gene), which are lin-53,pha-4,hmg-1.2, respectively. The human homologous gene FOXA1 of pha-4 plays an important role in the progression of prostate cancer in ruvb-1 and set-6,. In prostate cancer cells, EAF2 binds to FOXA1. Knockout of EAF2 in prostate cancer cell line LNCa P could up-regulate the protein level of endogenous FOXA1, while overexpression of EAF2 could decrease the expression of FOXA1. EAF2 had no effect on the expression of m RNA of FOXA1. Knockout of EAF2 expression in LNCa P cells can up-regulate the expression level of downstream AR gene, promote the binding of AR to ARE, and enhance cell proliferation and migration. Double knockout of FOXA1 and EAF2 could counteract the promotion of AR downstream gene expression by single knockout EAF2 and the proliferation and migration of, LNCa P cells combined with AR and ARE. It is suggested that FOXA1 mediates the regulation of AR transcription activity, cell proliferation and migration by EAF2. Conclusion: FOXA1 plays an important role in the regulation of AR signaling pathway, cell proliferation and migration in prostate cancer cells by EAF2.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25
本文编号：2424294
[Abstract]:Objective: ELL- related factor 2 (EAF2) is an important tumor suppressor in prostate cancer. The interaction between proteins is one of the important ways to regulate its function. The aim of this study was to study the molecular mechanism of EAF2 regulating androgen receptor activity and proliferation and migration of prostate cancer cells by screening and identifying proteins interacting with EAF2 in prostate cancer. Methods: the C.elegans model was used to screen RNAi and search for genes that could play a synergistic role with eaf-1. The binding of EAF2 to screened proteins was verified by immunoprecipitation assay. The regulation and mechanism of EAF2 on the expression of selected proteins were studied by realtime PCR and dose-dependent co-expression experiments. Si RNA gene interference technique was used to detect the expression level of AR downstream gene in prostate cancer cells by single knockout or double knockout, realtime PCR and double luciferase reporter gene in order to clarify the transcriptional activity of AR. Effect of EAF2 and screening protein interaction on the binding ability of AR to downstream gene DNA by chromatin immunoprecipitation assay; Brd U, clone formation assay and transwell cell migration assay were used to detect the changes of cell proliferation and migration ability. Results: using nematode fertility as a reference index, we screened out five genes that have synergistic effect with eaf-1 (C.elegans homologous gene of human EAF2 gene), which are lin-53,pha-4,hmg-1.2, respectively. The human homologous gene FOXA1 of pha-4 plays an important role in the progression of prostate cancer in ruvb-1 and set-6,. In prostate cancer cells, EAF2 binds to FOXA1. Knockout of EAF2 in prostate cancer cell line LNCa P could up-regulate the protein level of endogenous FOXA1, while overexpression of EAF2 could decrease the expression of FOXA1. EAF2 had no effect on the expression of m RNA of FOXA1. Knockout of EAF2 expression in LNCa P cells can up-regulate the expression level of downstream AR gene, promote the binding of AR to ARE, and enhance cell proliferation and migration. Double knockout of FOXA1 and EAF2 could counteract the promotion of AR downstream gene expression by single knockout EAF2 and the proliferation and migration of, LNCa P cells combined with AR and ARE. It is suggested that FOXA1 mediates the regulation of AR transcription activity, cell proliferation and migration by EAF2. Conclusion: FOXA1 plays an important role in the regulation of AR signaling pathway, cell proliferation and migration in prostate cancer cells by EAF2.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.25
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