本文選題：EB病毒(EBV) + 潛伏膜蛋白1(LMP1)　； 參考：《中南大學(xué)》2010年碩士論文

【摘要】： 真核生物基因的表達(dá)調(diào)控主要是轉(zhuǎn)錄水平的調(diào)控,其受順式作用元件與反式作用因子及其相互作用調(diào)節(jié),兩者結(jié)合才能共同實(shí)現(xiàn)對(duì)轉(zhuǎn)錄的調(diào)控。真核生物基因順式作用元件分為啟動(dòng)子、增強(qiáng)子、沉默子,是調(diào)節(jié)基因轉(zhuǎn)錄的特定DNA序列；參與調(diào)節(jié)靶基因轉(zhuǎn)錄的的反式作用因子(也稱為轉(zhuǎn)錄因子)是能直接或間接識(shí)別或結(jié)合在各順式作用元件核心序列上,參與調(diào)控轉(zhuǎn)錄效率的一類蛋白質(zhì)。 表皮生長因子受體(epidermal growth factor receptor, EGFR)是一個(gè)分子質(zhì)量為170kD的跨膜糖蛋白,屬于受體酪氨酸激酶(receptor tyrosine kinases, RTKs)家族。近年來,在許多腫瘤如皮膚癌、乳腺癌、宮頸癌、膀胱癌、卵巢癌等的細(xì)胞核內(nèi)發(fā)現(xiàn)高水平的EGFR表達(dá),其作為一種新型轉(zhuǎn)錄因子在核內(nèi)獨(dú)立或作為轉(zhuǎn)錄共活化子,與其他轉(zhuǎn)錄因子相互作用于與細(xì)胞周期行進(jìn)或細(xì)胞增殖密切相關(guān)的靶基因,促進(jìn)腫瘤的發(fā)生發(fā)展。轉(zhuǎn)錄因子STAT3(signal transducer and activation of transcription, STAT3)是多種細(xì)胞因子和生長因子發(fā)揮作用的關(guān)鍵信號(hào)分子,其通過酪氨酸磷酸化的STAT之間形成同源二聚體或異源二聚體活化后移位至細(xì)胞核,激活多種相關(guān)下游靶基因的表達(dá)。STAT3在腫瘤的發(fā)生發(fā)展中起促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡、免疫逃避、促進(jìn)血管生成及腫瘤細(xì)胞侵襲和轉(zhuǎn)移等多種生物學(xué)功能的作用。 EB病毒(Epstein-Barr Virus, EBV)是一種DNA致瘤病毒,與B細(xì)胞、上皮細(xì)胞、T細(xì)胞的惡性轉(zhuǎn)化有關(guān),并與多種腫瘤的發(fā)生發(fā)展相關(guān),其致瘤作用主要是通過病毒編碼蛋白影響宿主細(xì)胞的基因表達(dá)及相應(yīng)的生物學(xué)行為實(shí)現(xiàn)。潛伏膜蛋白1(Latent Membrane Protein1,LMP1)是EBV編碼的癌蛋白,其生物學(xué)功能涉及細(xì)胞轉(zhuǎn)化、凋亡、分化、細(xì)胞周期行進(jìn)以及參與惡性腫瘤的侵襲和轉(zhuǎn)移。 有研究發(fā)現(xiàn)在鼻咽癌細(xì)胞中LMP1能調(diào)控EGFR表達(dá)和上調(diào)EGFR磷酸化水平,進(jìn)而促進(jìn)細(xì)胞增殖,并發(fā)現(xiàn)LMP1可以調(diào)控EGFR核移位,并呈EGF配體非依賴性。同時(shí),不少作者認(rèn)為EGFR需與其他能直接結(jié)合于DNA的轉(zhuǎn)錄因子相互作用方能發(fā)揮促進(jìn)細(xì)胞增殖的生物學(xué)作用。并有報(bào)道發(fā)現(xiàn)在鼻咽癌細(xì)胞中存在LMP1激活的JAK/STAT信號(hào)通路,通過JAK3促進(jìn)STAT3 705位酪氨酸磷酸化,磷酸化活化后的STAT3即核移位并核內(nèi)積聚,并且LMP1調(diào)控磷酸化STAT3核移位呈705位酪氨酸磷酸化依賴性。因此,我們推測(cè)LMP1有可能通過誘導(dǎo)轉(zhuǎn)錄因子EGFR和STAT3核移位并調(diào)節(jié)其相互作用,從而在腫瘤發(fā)生發(fā)展中發(fā)揮新的生物學(xué)作用。 為觀察在LMP1調(diào)控下轉(zhuǎn)錄因子EGFR和STAT3在鼻咽癌細(xì)胞核內(nèi)是否存在核移位和共定位,選擇激光共聚焦掃描顯微鏡方法進(jìn)行檢測(cè),以不表達(dá)LMP1的鼻咽癌CNE1細(xì)胞為陰性對(duì)照,發(fā)現(xiàn)在LMP1陽性表達(dá)的鼻咽癌CNE1-LMP1細(xì)胞中存在EGFR和STAT3蛋白的核移位并且兩者存在共定位；應(yīng)用免疫共沉淀及Western-blot方法檢測(cè),顯示LMP1可促進(jìn)鼻咽癌細(xì)胞內(nèi)轉(zhuǎn)錄因子EGFR和STAT3呈復(fù)合物結(jié)合；為明確兩者復(fù)合物結(jié)合的亞細(xì)胞定位,分別提取了胞漿和核蛋白,進(jìn)一步應(yīng)用免疫共沉淀及Western-blot方法檢測(cè),揭示了EGFR和STAT3復(fù)合物結(jié)合的亞細(xì)胞定位主要是細(xì)胞核。 綜上所述,本研究發(fā)現(xiàn)了LMP1表達(dá)陽性的鼻咽癌細(xì)胞內(nèi)存在轉(zhuǎn)錄因子EGFR和STAT3的核移位和共定位,LMP1可調(diào)節(jié)EGFR和STAT3呈復(fù)合物結(jié)合并且其結(jié)合的亞細(xì)胞定位主要為細(xì)胞核。這一新發(fā)現(xiàn)從蛋白質(zhì)間相互作用的新視野,對(duì)EB病毒編碼的重要瘤蛋白LMP1調(diào)控轉(zhuǎn)錄因子EGFR和STAT3兩條不同的信號(hào)通路進(jìn)行了整合,從而對(duì)LMP1的功能進(jìn)行了拓展,同時(shí)為鼻咽癌分子靶向治療研發(fā)新的靶點(diǎn)提供了初步實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.63

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