LMP1调控转录因子EGFR和STAT3在鼻咽癌细胞核相互作用的研究

发布时间：2018-06-03 22:33

本文选题：EB病毒(EBV) + 潜伏膜蛋白1(LMP1)　；参考：《中南大学》2010年硕士论文

【摘要】： 真核生物基因的表达调控主要是转录水平的调控,其受顺式作用元件与反式作用因子及其相互作用调节,两者结合才能共同实现对转录的调控。真核生物基因顺式作用元件分为启动子、增强子、沉默子,是调节基因转录的特定DNA序列；参与调节靶基因转录的的反式作用因子(也称为转录因子)是能直接或间接识别或结合在各顺式作用元件核心序列上,参与调控转录效率的一类蛋白质。表皮生长因子受体(epidermal growth factor receptor, EGFR)是一个分子质量为170kD的跨膜糖蛋白,属于受体酪氨酸激酶(receptor tyrosine kinases, RTKs)家族。近年来,在许多肿瘤如皮肤癌、乳腺癌、宫颈癌、膀胱癌、卵巢癌等的细胞核内发现高水平的EGFR表达,其作为一种新型转录因子在核内独立或作为转录共活化子,与其他转录因子相互作用于与细胞周期行进或细胞增殖密切相关的靶基因,促进肿瘤的发生发展。转录因子STAT3(signal transducer and activation of transcription, STAT3)是多种细胞因子和生长因子发挥作用的关键信号分子,其通过酪氨酸磷酸化的STAT之间形成同源二聚体或异源二聚体活化后移位至细胞核,激活多种相关下游靶基因的表达。STAT3在肿瘤的发生发展中起促进细胞增殖、抑制细胞凋亡、免疫逃避、促进血管生成及肿瘤细胞侵袭和转移等多种生物学功能的作用。 EB病毒(Epstein-Barr Virus, EBV)是一种DNA致瘤病毒,与B细胞、上皮细胞、T细胞的恶性转化有关,并与多种肿瘤的发生发展相关,其致瘤作用主要是通过病毒编码蛋白影响宿主细胞的基因表达及相应的生物学行为实现。潜伏膜蛋白1(Latent Membrane Protein1,LMP1)是EBV编码的癌蛋白,其生物学功能涉及细胞转化、凋亡、分化、细胞周期行进以及参与恶性肿瘤的侵袭和转移。有研究发现在鼻咽癌细胞中LMP1能调控EGFR表达和上调EGFR磷酸化水平,进而促进细胞增殖,并发现LMP1可以调控EGFR核移位,并呈EGF配体非依赖性。同时,不少作者认为EGFR需与其他能直接结合于DNA的转录因子相互作用方能发挥促进细胞增殖的生物学作用。并有报道发现在鼻咽癌细胞中存在LMP1激活的JAK/STAT信号通路,通过JAK3促进STAT3 705位酪氨酸磷酸化,磷酸化活化后的STAT3即核移位并核内积聚,并且LMP1调控磷酸化STAT3核移位呈705位酪氨酸磷酸化依赖性。因此,我们推测LMP1有可能通过诱导转录因子EGFR和STAT3核移位并调节其相互作用,从而在肿瘤发生发展中发挥新的生物学作用。为观察在LMP1调控下转录因子EGFR和STAT3在鼻咽癌细胞核内是否存在核移位和共定位,选择激光共聚焦扫描显微镜方法进行检测,以不表达LMP1的鼻咽癌CNE1细胞为阴性对照,发现在LMP1阳性表达的鼻咽癌CNE1-LMP1细胞中存在EGFR和STAT3蛋白的核移位并且两者存在共定位；应用免疫共沉淀及Western-blot方法检测,显示LMP1可促进鼻咽癌细胞内转录因子EGFR和STAT3呈复合物结合；为明确两者复合物结合的亚细胞定位,分别提取了胞浆和核蛋白,进一步应用免疫共沉淀及Western-blot方法检测,揭示了EGFR和STAT3复合物结合的亚细胞定位主要是细胞核。综上所述,本研究发现了LMP1表达阳性的鼻咽癌细胞内存在转录因子EGFR和STAT3的核移位和共定位,LMP1可调节EGFR和STAT3呈复合物结合并且其结合的亚细胞定位主要为细胞核。这一新发现从蛋白质间相互作用的新视野,对EB病毒编码的重要瘤蛋白LMP1调控转录因子EGFR和STAT3两条不同的信号通路进行了整合,从而对LMP1的功能进行了拓展,同时为鼻咽癌分子靶向治疗研发新的靶点提供了初步实验依据。
[Abstract]:The regulation of eukaryotic gene expression is mainly regulated by the transcription level, which is regulated by the cis acting element and the trans acting factor and their interaction. The cis acting elements of eukaryotes can be divided into promoters, enhancers and silencers, which are the specific DNA sequences that regulate gene transcription. Trans acting factors (also known as transcription factors) involved in the regulation of target gene transcription are a class of proteins that can directly or indirectly identify or bind to the core sequences of various cis acting elements and participate in the regulation of transcriptional efficiency.
Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein with a molecular mass of 170kD, belonging to the receptor tyrosine kinase (receptor tyrosine kinases, RTKs) family. In recent years, high level EGFR tables have been found in many tumors, such as skin cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer and so on. The transcription factor STAT3 (signal transducer and activation of transcription, STAT3) is a new type of transcriptional factor that is independent or transcriptional co activator, interacting with other transcription factors in a target gene closely related to cell cycle progression or cell proliferation. The transcription factor (transducer and activation of transcription, STAT3) is a variety of cellular causes. The key signaling molecules that play a role in the growth factor and growth factor are activated by the activation of a homologous two polymer or heterogenous two polymer between the tyrosine phosphorylated STAT and the activation of a variety of related downstream target genes to stimulate the proliferation of cells, inhibit cell apoptosis, immune escape, and promote blood in the development of tumor. Guan Shengcheng and tumor cell invasion and metastasis and other biological functions.
The EB virus (Epstein-Barr Virus, EBV) is a DNA oncovirus, which is related to the malignant transformation of B cells, epithelial cells and T cells, and is related to the development of a variety of tumors. The tumorigenesis is mainly caused by the effect of virus encoded protein on the gene expression of host cells and the biological behavior of the host cells. Latent membrane protein 1 (Latent Membrane). Protein1, LMP1) is a EBV encoded oncoprotein, whose biological functions involve cell transformation, apoptosis, differentiation, cell cycle progression, and involvement in the invasion and metastasis of malignant tumors.
Some studies have found that in nasopharyngeal carcinoma cells LMP1 can regulate the expression of EGFR and up the level of EGFR phosphorylation, and then promote cell proliferation, and it is found that LMP1 can regulate the EGFR nuclear shift and is not dependent on the EGF ligand. At the same time, many authors believe that EGFR needs to play a role in promoting cell proliferation with other transcription factors that can be directly combined with DNA. It is reported that there is a LMP1 activated JAK/STAT signaling pathway in nasopharyngeal carcinoma cells, which promotes STAT3 705 tyrosine phosphorylation through JAK3, STAT3 after phosphorylation activation and accumulation in the nucleus, and LMP1 regulates the phosphorylation of STAT3 nucleus to 705 tyrosine phosphorylation dependence. Therefore, we speculate LMP1 It is possible to play a new biological role in tumor development by inducing the transcription factor EGFR and STAT3 nuclear to translocate and regulate their interactions.
In order to observe whether the transcription factor EGFR and STAT3 have nuclear shift and co location in the nucleus of nasopharyngeal carcinoma under the control of LMP1, the laser confocal scanning microscope was selected to detect the negative control of the nasopharyngeal carcinoma CNE1 cells that did not express LMP1, and the presence of EGFR and STAT3 protein in the LMP1 positive expression of nasopharyngeal carcinoma CNE1-LMP1 cells was found. Nuclear transfer and co localization were found. By using immunoprecipitation and Western-blot method, LMP1 could promote the combination of EGFR and STAT3 in nasopharyngeal carcinoma cells, and the cytoplasm and nuclear egg white were extracted for the subcellular localization of the combination of the two compounds, and the immunoprecipitation and Western-blot were further applied. Methods detection revealed that the subcellular localization of EGFR and STAT3 complex was mainly the nucleus.
To sum up, this study found the nuclear transfer and co localization of transcription factors EGFR and STAT3 in LMP1 positive nasopharyngeal carcinoma cells. LMP1 can regulate the combination of EGFR and STAT3 and its subcellular localization is mainly the nucleus. This new field of vision from interprotein interphase interaction is important for the importance of the encoding of EB virus. The tumor protein LMP1 regulates the two different signaling pathways of the transcription factor EGFR and STAT3, thereby expanding the function of LMP1 and providing a preliminary experimental basis for the development of new targets for the molecular targeting therapy of nasopharyngeal carcinoma.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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