【摘要】：目的:ELL-相關因子2(EAF2)是前列腺癌中重要的腫瘤抑制因子,然而其抑癌作用的具體機制尚不清楚。蛋白間的相互作用是調(diào)控其功能的重要方式之一。本研究擬通過篩選并鑒定前列腺癌中與EAF2相互作用的蛋白,探討EAF2調(diào)控雄激素受體活性及前列腺癌細胞增殖、遷移的分子機制。方法:應用線蟲(C.elegans)模型進行RNAi篩查,尋找與eaf-1發(fā)揮協(xié)同作用的基因。應用免疫共沉淀實驗驗證EAF2與篩選蛋白的結合情況。通過realtime PCR和劑量依賴共表達實驗研究EAF2對所選蛋白表達的調(diào)控作用及機制。利用si RNA基因干擾技術對前列腺癌細胞中EAF2和所選蛋白進行單獨敲除或雙敲除,realtime PCR和雙熒光素酶報告基因檢測AR下游基因的表達水平以明確AR轉(zhuǎn)錄活性變化;染色質(zhì)免疫共沉淀實驗檢測EAF2和篩選蛋白相互作用對AR與下游基因DNA結合能力的影響;Brd U、克隆形成實驗和transwell細胞遷移實驗檢測細胞增殖和遷移能力的改變。結果:以線蟲生育能力作為參考指標,我們篩選出5個與eaf-1(人類EAF2基因的C.elegans同源基因)具有協(xié)同作用的基因,分別為lin-53,pha-4,hmg-1.2,ruvb-1和set-6,其中pha-4的人類同源基因FOXA1在前列腺癌進展中具有重要作用,因此我們選擇其作為后續(xù)研究基因。在前列腺癌細胞中,EAF2與FOXA1相互結合。敲除前列腺癌細胞LNCa P中的EAF2能夠上調(diào)內(nèi)源性FOXA1的蛋白水平,過表達EAF2則能降低FOXA1的表達。EAF2對FOXA1的m RNA表達無影響。敲除LNCa P細胞的EAF2表達能夠上調(diào)AR下游基因表達水平,促進AR與ARE結合,并增強細胞的增殖和遷移能力;對FOXA1和EAF2進行雙敲除則能夠抵消單獨敲除EAF2對AR下游基因表達、AR同ARE的結合、LNCa P細胞增殖和遷移能力的促進作用。表明FOXA1在EAF2對AR轉(zhuǎn)錄活性、細胞增殖和遷移的調(diào)控中起介導作用。結論:在EAF2調(diào)控前列腺癌細胞AR信號通路、細胞增殖和遷移能力的過程中,FOXA1發(fā)揮了重要的介導作用。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.25

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