本文關(guān)鍵詞：EB病毒潛伏膜蛋白LMP1通過轉(zhuǎn)錄因子EGFR和共因子TIF2調(diào)控cyclinD1基因的機(jī)制研究　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： EB病毒 潛伏膜蛋白1 EGFR TIF2 cyclinD1

【摘要】：表皮生長因子受體(epidermal growth factor receptor, EGFR)屬于受體酪氨酸激酶家族成員,與腫瘤癌變密切相關(guān)。我們的前期研究證明在EB病毒(Epstein-B arr virus, EBV)編碼的潛伏膜蛋白LMP1(latent membrane protein1)調(diào)控下核EGFR可與cyclinD1啟動子結(jié)合,且其他的蛋白可能參與這一過程。轉(zhuǎn)錄中介因子(Transcriptional intermediary factor2, TIF2)作為一類輔激活因子除與核受體結(jié)合外還可與轉(zhuǎn)錄因子發(fā)生相互作用,同時它也被報道可參與cyclinDl基因轉(zhuǎn)錄的精細(xì)調(diào)控,從而提示我們EGFR與TIF2有可能存在一些新的直接聯(lián)系。因此本研究以鼻咽癌細(xì)胞為模型,以EBV LMP1激活轉(zhuǎn)錄因子EGFR和共因子TIF2相互作用調(diào)控細(xì)胞周期行進(jìn)為切入點,深入研究EBV LMP1致瘤的分子新機(jī)理。 我們以LMP1陰性和LMP1陽性表達(dá)的鼻咽癌細(xì)胞為模型,利用Real-time PCR. Western blotting、免疫共沉淀、雙雜交策略及激光共聚焦技術(shù),對EBV LMP1調(diào)控轉(zhuǎn)錄因子EGFR和共因子TIF2的轉(zhuǎn)錄活化及亞細(xì)胞定位進(jìn)行研究,發(fā)現(xiàn)鼻咽癌細(xì)胞中EBV LMP1可上調(diào)TIF2的蛋白表達(dá)并呈劑量依賴性,但對TIF2的mRNA水平?jīng)]有影響；確定了EBV LMP1調(diào)控EGFR和TIF2蛋白相互間形成復(fù)合物,且這種相互作用在LMP1的誘導(dǎo)下顯著增高,其亞細(xì)胞定位在細(xì)胞核。 進(jìn)一步為了探討LMP1激活EGFR和TIF2相互作用參與轉(zhuǎn)錄精細(xì)調(diào)控的分子機(jī)制,我們采用ChIP/Re-ChIP、報告基因分析和點突變技術(shù),結(jié)合過表達(dá)和RNA干擾手段,初步證實cyclinD1基因啟動子區(qū)存在EGFR和TIF2共結(jié)合位點；從轉(zhuǎn)錄水平、蛋白水平和mRNA水平確定EBV-LMP1可調(diào)控EGFR和TIF2轉(zhuǎn)錄活化靶基因cyclinD1,其可能的調(diào)節(jié)機(jī)制是LMP1促使EGFR結(jié)合于cyclinD1啟動子區(qū)并募集TIF2與其相互作用,激活cyclinD1基因轉(zhuǎn)錄；EGFR/TIF2復(fù)合物對cyclinD1啟動活性的影響主要依賴于基因啟動子區(qū)ATRS序列。 最后我們采用MTS方法,通過外源性的轉(zhuǎn)入EGFR/TIF2表達(dá)質(zhì)粒和內(nèi)源性的敲除EGFR/TIF2的表達(dá),證實了EBV LMP1通過調(diào)控EGFR和TIF2相互作用提高細(xì)胞的生存活性。接著采用流式細(xì)胞術(shù)結(jié)合PI染色法對CNE1/CNE1-LMP1細(xì)胞內(nèi)DNA含量進(jìn)行檢測,發(fā)現(xiàn)在EBVLMP1誘導(dǎo)情況下可促進(jìn)細(xì)胞通過G1/S期限制點,加速細(xì)胞不成熟地進(jìn)入S期,同時LMP1的表達(dá)可能會導(dǎo)致細(xì)胞阻滯于G2/M期。此外,結(jié)果還進(jìn)一步證實LMP1促進(jìn)細(xì)胞周期行進(jìn)是通過EGFR和TIF2相互作用來實現(xiàn)的。 本課題以鼻咽癌細(xì)胞為模型,首次觀察到LMP1可調(diào)控轉(zhuǎn)錄因子EGFR和共因子TIF2呈復(fù)合物形式共定位于細(xì)胞核；同樣首次發(fā)現(xiàn)LMP1能促使EGFR結(jié)合于cyclinD1啟動子區(qū)并募集TIF2與其相互作用,以一種合作的方式轉(zhuǎn)錄活化cyclinD1基因；其主要的生物學(xué)意義在于在加速G1/S期的進(jìn)程,從而影響細(xì)胞周期和細(xì)胞生存活性。這一新發(fā)現(xiàn)為核EGFR和共因子在細(xì)胞周期進(jìn)程中的作用提供了新的直接聯(lián)系,同時也為EB病毒致瘤分子機(jī)理研究開拓了新的視野。
[Abstract]:Epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family. Our previous studies have shown that Epstein-B arr virus is associated with Epstein-B arr virus. Under the regulation of LMP1(latent membrane protein 1, a latent membrane protein encoded by EBV, nuclear EGFR binds to cyclinD1 promoter. And other proteins may be involved in this process. Transcriptional intermediary factor2. As a kind of coactivator, TIF2 can interact with transcription factors in addition to binding to nuclear receptors. It has also been reported that TIF2 can be involved in the fine regulation of cyclinDl gene transcription. It suggests that there may be some new direct relationship between EGFR and TIF2. Therefore, this study is based on nasopharyngeal carcinoma cell model. Based on the interaction of EBV LMP1 activated transcription factor EGFR and cofactor TIF2 to regulate cell cycle progression, the molecular mechanism of EBV LMP1 tumorigenesis was studied. We used LMP1 negative and LMP1 positive nasopharyngeal carcinoma cells as a model, using Real-time PCR. Western blotting, immunoprecipitation. The transcriptional activation and subcellular localization of transcription factor EGFR and cofactor TIF2 regulated by EBV LMP1 were studied by two-hybrid strategy and laser confocal technique. It was found that EBV LMP1 could up-regulate the expression of TIF2 protein in a dose-dependent manner in nasopharyngeal carcinoma cells, but had no effect on the mRNA level of TIF2. It is confirmed that EBV LMP1 regulates the formation of complexes between EGFR and TIF2 proteins, and this interaction is significantly increased under the induction of LMP1, and its subcellular localization is located in the nucleus. In order to explore the molecular mechanism of LMP1 activating the interaction between EGFR and TIF2 involved in the fine regulation of transcription, we used ChIP/Re-ChIP. The co-binding sites of EGFR and TIF2 in the promoter region of cyclinD1 gene were preliminarily confirmed by reporter gene analysis and point mutation technique combined with overexpression and RNA interference. From the transcriptional level, protein level and mRNA level, EBV-LMP1 can regulate the transcriptional activation target gene cyclinD1 of EGFR and TIF2. The possible regulatory mechanism is that LMP1 stimulates EGFR to bind to the cyclinD1 promoter and to recruit TIF2 to interact with it to activate cyclinD1 gene transcription. The effect of EGFR/TIF2 complex on cyclinD1 promoter activity mainly depends on the ATRS sequence of gene promoter region. Finally, we use MTS method, through exogenous transfer of EGFR/TIF2 expression plasmid and endogenous knockout EGFR/TIF2 expression. Confirmed EBV. LMP1 enhanced the viability of CNE1/CNE1-LMP1 cells by regulating the interaction between EGFR and TIF2. Then, the content of DNA in CNE1/CNE1-LMP1 cells was determined by flow cytometry and Pi staining. For testing. It was found that under the condition of EBVLMP1 induction, cells could be accelerated to enter S phase immature through G 1 / S phase restriction point. The expression of LMP1 may lead to cell arrest in G _ 2 / M phase. Furthermore, it is further demonstrated that LMP1 promotes cell cycle progression through the interaction of EGFR and TIF2. In this study, we first observed that LMP1 can regulate transcription factor EGFR and cofactor TIF2 to co-locate in the nucleus of nasopharyngeal carcinoma cells in the form of complex. It is also the first time that LMP1 can induce EGFR to bind to the promoter of cyclinD1 and recruit TIF2 to interact with it to transcribe and activate cyclinD1 gene in a cooperative manner. Its main biological significance lies in accelerating the process of G1 / S phase. This new discovery provides a new direct link between nuclear EGFR and co-factors in the process of cell cycle. At the same time, it also opens up a new field of vision for the study of Epstein-Barr virus tumorigenic molecular mechanism.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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