本文選題：DEC1 + cyclin��； 參考：《大連理工大學(xué)》2015年博士論文

【摘要】：全球每年都有數(shù)百萬人死于癌癥,癌癥亦稱惡性腫瘤,它的無限制、無止境地增生往往與細(xì)胞周期紊亂有關(guān)。越來越多的數(shù)據(jù)表明細(xì)胞周期蛋白cyclin E過表達(dá)或其基因擴增與腫瘤形成密切相關(guān)。cyclin E的表達(dá)受多種因子的調(diào)節(jié),這些因子通過調(diào)節(jié)cyclinE的轉(zhuǎn)錄、翻譯及翻譯后修飾進而影響腫瘤的發(fā)生發(fā)展。時鐘蛋白DEC1作為一個重要的轉(zhuǎn)錄因子,可以調(diào)節(jié)多種靶基因的表達(dá),參與細(xì)胞分化、凋亡、衰老等生物學(xué)過程進而調(diào)控腫瘤的發(fā)生發(fā)展。目前的研究結(jié)果表明DEC1既可以抑制腫瘤的生長、介導(dǎo)p53誘導(dǎo)的細(xì)胞衰老,又可以促進腫瘤細(xì)胞的生存起到抗凋亡的作用,但是DEC1如何調(diào)節(jié)腫瘤細(xì)胞生存以及細(xì)胞周期進程的分子機理還不清楚。因此本文著重研究了DEC1如何影響乳腺癌細(xì)胞的增殖以及對細(xì)胞周期蛋白cyclin E穩(wěn)定性及功能的影響。本文主要工作如下：1.通過克隆形成實驗和MTT實驗發(fā)現(xiàn)DEC1的過表達(dá)抑制了MCF-7和T47D細(xì)胞的生長。相反,干擾DEC1后明顯抑制了細(xì)胞集落的大小和數(shù)量。2.在MCF-7細(xì)胞中通過免疫印跡、流式細(xì)胞術(shù)等方法研究DEC1表達(dá)的變化。實驗發(fā)現(xiàn)時鐘蛋白DEC1在細(xì)胞周期的進程中其自身表達(dá)存在周期性,主要在G1/S期表達(dá)量較高,其表達(dá)的變化與細(xì)胞周期蛋白cyclin E相似。在MCF-7和T47D兩種乳腺癌細(xì)胞中,DEC1以劑量依賴的方式上調(diào)cyclin E蛋白含量,并且不影響cyclin E的mRNA水平。3.DEC1上調(diào)cyclin E的蛋白含量但不影響其轉(zhuǎn)錄,因此可能影響cyclin E的降解。通過半衰期實驗證實DEC1穩(wěn)定了cyclin E,延長了cyclin E的半衰期。通過免疫印跡實驗,發(fā)現(xiàn)DEC1對cyclin E的調(diào)節(jié)依賴cyclin E的泛素E3酶Fbw7,通過免疫共沉淀實驗證實DEC1抑制cyclin E與Fbw7a相互作用,抑制cyclin E的泛素化,進而穩(wěn)定cyclinE。4.通過免疫共沉淀實驗,哺乳動物雙雜交實驗證實在血清饑餓及正常條件下DEC1與周期蛋白cyclin E存在相互作用。同時,內(nèi)源的實驗也驗證了DEC1與cyclin E存在相互作用。通過熒光共定位實驗發(fā)現(xiàn),cyclin E與DEC1很好的定位在細(xì)胞核中。另外,檢測了DEC1與cyclin E相互作用動力學(xué),結(jié)果發(fā)現(xiàn)DEC1與cyclin E主要是在G1及S期存在相互作用,在恢復(fù)培養(yǎng)8h后其相互作用最明顯,即G1/S期檢測點。5.研究DEC1對cyclin E功能及細(xì)胞周期進程的影響。cyclin E通常與Cdk2相互作用之后被降解,實現(xiàn)cyclin A與Cdk2的結(jié)合。通過共沉淀結(jié)果發(fā)現(xiàn)DEC1增加了cyclinE/Cdk2,抑制了隨后的cyclin A/Cdk2的結(jié)合,引起細(xì)胞S期延長,導(dǎo)致細(xì)胞周期停滯。最后在裸鼠腫瘤實驗中發(fā)現(xiàn)DEC1明顯抑制了腫瘤的生長。本論文的研究將時鐘蛋白DEC1與細(xì)胞周期因子cyclin E聯(lián)系起來,確定DEC1抑制cyclin E泛素化降解過程影響了cyclin E的功能,進而抑制乳腺癌細(xì)胞的增殖,本研究為乳腺癌的治療,特別是cyclin E高表達(dá)的乳腺癌的治療提供理論依據(jù),為選擇特異性藥物靶點、優(yōu)化治療措施提供科學(xué)基礎(chǔ)。
[Abstract]:Millions of people worldwide die each year from cancer, also known as a malignant tumor, whose unlimited, never-ending proliferation is often associated with cell cycle disorders. More and more data show that the overexpression of cyclin E or its gene amplification is closely related to tumor formation, and the expression of cyclin E is regulated by a variety of factors, which regulate the transcription of cyclin E. Translation and posttranslational modification affect the development of tumor. As an important transcription factor, clock protein DEC1 can regulate the expression of multiple target genes, participate in the biological processes of cell differentiation, apoptosis and senescence, and then regulate the occurrence and development of tumor. The present results indicate that DEC1 can not only inhibit tumor growth, mediate p53 induced cell senescence, but also promote the survival of tumor cells and play an anti-apoptotic role. However, the molecular mechanism of DEC1 regulating tumor cell survival and cell cycle progression is unclear. In this paper, we focus on the effects of Decl on the proliferation of breast cancer cells and on the stability and function of cyclin E. The main work of this paper is as follows: 1. The overexpression of DEC1 inhibited the growth of MCF-7 and T47D cells by clone formation assay and MTT assay. In contrast, interference with DEC1 significantly inhibited the size and number of cell colonies. The changes of DEC1 expression in MCF-7 cells were studied by Western blotting and flow cytometry. It was found that the clock protein DEC1 was cyclically expressed in the process of cell cycle, mainly in the G 1 / S phase, and the change of its expression was similar to that of cyclin E. In MCF-7 and T47D breast cancer cells, DEC1 up-regulated the cyclin E protein content in a dose-dependent manner, and did not affect the cyclin E mRNA level. 3. DEC1 upregulated the cyclin E protein content but did not affect its transcription, so it may affect the degradation of cyclin E. The half-life experiment proved that Decl stabilized cyclin E and prolonged the half life of cyclin E. By immunoblotting, we found that the regulation of DEC1 on cyclin E was dependent on the ubiquitin E3 enzyme Fbw7 of cyclin E. By immunoprecipitation, it was proved that Dec1 inhibited the interaction between cyclin E and Fbw7a, inhibited the ubiquification of cyclin E, and then stabilized cyclin E. 4. The interaction between DEC1 and cyclin E was confirmed by immunoprecipitation assay and mammalian two-hybrid assay under the condition of serum starvation and normal conditions. At the same time, the interaction between DEC1 and cyclin E was verified by endogenous experiments. It was found that cyclin E and DEC1 were well located in the nucleus. In addition, the kinetics of interaction between DEC1 and cyclin E was detected. It was found that the interaction between DEC1 and cyclin E was mainly in G1 and S phase, and the interaction was the most obvious after 8 h of recovery culture, that is, G1 / S phase detection point. Study on the effect of DEC1 on cyclin E function and cell cycle progression. Cyclin E is usually degraded after interaction with Cdk2 to achieve the binding of cyclin A and Cdk2. The results of coprecipitation showed that DEC1 increased cyclin E / CD ~ (2), inhibited the subsequent binding of cyclin A / CD ~ (2), prolonged S phase and led to cell cycle arrest. At last, DEC1 was found to inhibit tumor growth in nude mice. In this study, the clock protein DEC1 was associated with the cell cycle factor cyclin E, and it was concluded that Decl inhibited the degradation of cyclin E by affecting the function of cyclin E and thus inhibited the proliferation of breast cancer cells. In particular, the treatment of breast cancer with high expression of cyclin E provides a theoretical basis for the selection of specific drug targets and the optimization of treatment measures.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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