EB病毒潜伏膜蛋白LMP1通过转录因子EGFR和共因子TIF2调控cyclinD1基因的机制研究

发布时间：2018-01-16 18:45

本文关键词：EB病毒潜伏膜蛋白LMP1通过转录因子EGFR和共因子TIF2调控cyclinD1基因的机制研究　出处：《中南大学》2012年硕士论文　论文类型：学位论文

更多相关文章： EB病毒 潜伏膜蛋白1 EGFR TIF2 cyclinD1

【摘要】：表皮生长因子受体(epidermal growth factor receptor, EGFR)属于受体酪氨酸激酶家族成员,与肿瘤癌变密切相关。我们的前期研究证明在EB病毒(Epstein-B arr virus, EBV)编码的潜伏膜蛋白LMP1(latent membrane protein1)调控下核EGFR可与cyclinD1启动子结合,且其他的蛋白可能参与这一过程。转录中介因子(Transcriptional intermediary factor2, TIF2)作为一类辅激活因子除与核受体结合外还可与转录因子发生相互作用,同时它也被报道可参与cyclinDl基因转录的精细调控,从而提示我们EGFR与TIF2有可能存在一些新的直接联系。因此本研究以鼻咽癌细胞为模型,以EBV LMP1激活转录因子EGFR和共因子TIF2相互作用调控细胞周期行进为切入点,深入研究EBV LMP1致瘤的分子新机理。我们以LMP1阴性和LMP1阳性表达的鼻咽癌细胞为模型,利用Real-time PCR. Western blotting、免疫共沉淀、双杂交策略及激光共聚焦技术,对EBV LMP1调控转录因子EGFR和共因子TIF2的转录活化及亚细胞定位进行研究,发现鼻咽癌细胞中EBV LMP1可上调TIF2的蛋白表达并呈剂量依赖性,但对TIF2的mRNA水平没有影响；确定了EBV LMP1调控EGFR和TIF2蛋白相互间形成复合物,且这种相互作用在LMP1的诱导下显著增高,其亚细胞定位在细胞核。进一步为了探讨LMP1激活EGFR和TIF2相互作用参与转录精细调控的分子机制,我们采用ChIP/Re-ChIP、报告基因分析和点突变技术,结合过表达和RNA干扰手段,初步证实cyclinD1基因启动子区存在EGFR和TIF2共结合位点；从转录水平、蛋白水平和mRNA水平确定EBV-LMP1可调控EGFR和TIF2转录活化靶基因cyclinD1,其可能的调节机制是LMP1促使EGFR结合于cyclinD1启动子区并募集TIF2与其相互作用,激活cyclinD1基因转录；EGFR/TIF2复合物对cyclinD1启动活性的影响主要依赖于基因启动子区ATRS序列。最后我们采用MTS方法,通过外源性的转入EGFR/TIF2表达质粒和内源性的敲除EGFR/TIF2的表达,证实了EBV LMP1通过调控EGFR和TIF2相互作用提高细胞的生存活性。接着采用流式细胞术结合PI染色法对CNE1/CNE1-LMP1细胞内DNA含量进行检测,发现在EBVLMP1诱导情况下可促进细胞通过G1/S期限制点,加速细胞不成熟地进入S期,同时LMP1的表达可能会导致细胞阻滞于G2/M期。此外,结果还进一步证实LMP1促进细胞周期行进是通过EGFR和TIF2相互作用来实现的。本课题以鼻咽癌细胞为模型,首次观察到LMP1可调控转录因子EGFR和共因子TIF2呈复合物形式共定位于细胞核；同样首次发现LMP1能促使EGFR结合于cyclinD1启动子区并募集TIF2与其相互作用,以一种合作的方式转录活化cyclinD1基因；其主要的生物学意义在于在加速G1/S期的进程,从而影响细胞周期和细胞生存活性。这一新发现为核EGFR和共因子在细胞周期进程中的作用提供了新的直接联系,同时也为EB病毒致瘤分子机理研究开拓了新的视野。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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