本文選題：雌激素受體 + 雌激素(E2)　； 參考：《大連理工大學(xué)》2012年博士論文

【摘要】：周期節(jié)律的破壞與多種激素相關(guān)的腫瘤的發(fā)生有著密切的關(guān)系,如乳腺癌的發(fā)生,而雌激素受體ER信號(hào)通路的異常是乳腺癌發(fā)生的一個(gè)重要誘導(dǎo)因素,但周期節(jié)律的破壞與腫瘤發(fā)生發(fā)展間的分子機(jī)理還不清楚。核心時(shí)鐘蛋白CLOCK (circadian locomoter activity kaput)是一種堿性螺旋-環(huán)-螺旋(basic helix-loop-helix/Per-Arnt-Sim homology domain, bHLH/PAS)蛋白,含有bHLH/PAS結(jié)構(gòu)域。在周期節(jié)律中,CLOCK通常與BMAL1形成異源二聚體以調(diào)控下游基因的轉(zhuǎn)錄,這在周期節(jié)律的維持上發(fā)揮重要作用。但目前對(duì)于CLOCK是否直接參與到腫瘤相關(guān)通路中的研究還很少。 蛋白的翻譯后修飾包括磷酸化,乙�；�,SUMO化,泛素化等,這些翻譯后修飾對(duì)于調(diào)控蛋白的功能發(fā)揮重要作用。目前對(duì)于CLOCK的翻譯后修飾的研究還很少,僅發(fā)現(xiàn)其可被PKC (protein kinase C)和GSK-3(glycogen synthase kinase-3)磷酸化修飾。本研究的另一著眼點(diǎn)即CLOCK的SUMO化修飾研究,以及SUMO化修飾對(duì)其功能影響,尤其是對(duì)雌激素信號(hào)通路的影響。本研究以雌激素受體ERa陽(yáng)性的MCF-7細(xì)胞為研究對(duì)象,主要工作如下： 1.在MCF-7細(xì)胞中發(fā)現(xiàn)時(shí)鐘蛋白CLOCK與雌激素受體ERa間存在相互作用,并且通過(guò)雌激素(E2)刺激,可增強(qiáng)二者的相互作用,通過(guò)報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn)在雌激素存在的情況下,CLOCK對(duì)ERa的轉(zhuǎn)錄活性有明顯的促進(jìn)作用,說(shuō)明CLOCK可能作為ERa的輔激活因子,參與了由ERa介導(dǎo)的雌激素信號(hào)通路。 2.通過(guò)結(jié)構(gòu)及序列分析發(fā)現(xiàn)CLOCK有2個(gè)潛在的SUMO化修飾位點(diǎn),即K67和K851,表明CLOCK可能是一個(gè)SUMO修飾底物。隨后,CLOCK在MCF-7細(xì)胞中被證明可以被SUMO化,同時(shí)CLOCK的SUMO化在E2刺激的條件下有增強(qiáng)的趨勢(shì),通過(guò)定點(diǎn)突變,構(gòu)建了3個(gè)突變體,即CLOCK K67R, K851R, K67R/K851R (2K/2R)。單位點(diǎn)突變(K67R或K851R)與野生型相比,使得CLOCK的SUMO化水平下降,而雙位點(diǎn)突變體2K/2R使得CLOCK的SUMO化消失,并降低其自身轉(zhuǎn)錄活性。通過(guò)使用SENPs進(jìn)行去SUMO化研究發(fā)現(xiàn),SENP1作為CLOCK的去SUMO化酶介導(dǎo)了CLOCK的去SUMO化,并降低了CLOCK的轉(zhuǎn)錄活性。 3.通過(guò)核質(zhì)分離及免疫熒光實(shí)驗(yàn),發(fā)現(xiàn)SUMO化CLOCK主要分布在細(xì)胞核中,而去SUMO化減弱了CLOCK在細(xì)胞核中的分布,進(jìn)一步研究發(fā)現(xiàn)CLOCK的SUMO化雙位點(diǎn)突變體,即CLOCK2K/2R,減弱了CLOCK與BMAL1間的相互作用,從而降低了CLOCK的轉(zhuǎn)錄活性及在細(xì)胞核中的定位。 4. SUMO化修飾通常會(huì)改變蛋白的結(jié)構(gòu),從而影響了底物蛋白與其他蛋白間的相互作用。通過(guò)免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn),去SUMO化的CLOCK減弱了CLOCK與ERα間的相互作用。接下來(lái),通過(guò)報(bào)告基因?qū)嶒?yàn)發(fā)現(xiàn)SUMO化CLOCK可增強(qiáng)ERα轉(zhuǎn)錄活性,因?yàn)橐吧虲LOCK的過(guò)表達(dá)可明顯激活ERα的轉(zhuǎn)錄活性,而SUMO化位點(diǎn)突變的CLOCK,即CLOCK2K/2R卻喪失了這一功能。 5. Cyclin D1是ERα的經(jīng)典下游靶基因,并且對(duì)細(xì)胞增殖有促進(jìn)作用。通過(guò)實(shí)時(shí)定量PCR及報(bào)告基因?qū)嶒?yàn)研究發(fā)現(xiàn),野生型CLOCK能夠激活Cyclin D1的表達(dá),而CLOCK2K/2R卻不能。同時(shí),通過(guò)MTT對(duì)細(xì)胞增殖情況進(jìn)行研究發(fā)現(xiàn),野生型CLOCK能促進(jìn)MCF-7細(xì)胞的增殖,流式細(xì)胞實(shí)驗(yàn)也得出類(lèi)似的結(jié)果,即野生型CLOCK的過(guò)表達(dá)降低了G0/G1期細(xì)胞數(shù)量,相應(yīng)的提高了S期細(xì)胞數(shù)量,而CLOCK的雙突變體的過(guò)表達(dá)對(duì)細(xì)胞周期的影響不大。 通過(guò)本論文的研究將時(shí)鐘蛋白CLOCK與ERa介導(dǎo)的雌激素信號(hào)通路聯(lián)系起來(lái),并確定了CLOCK的一種重要翻譯后修飾,SUMO化。值得注意的是,SUMO化修飾對(duì)于CLOCK在雌激素信號(hào)通路的調(diào)控中發(fā)揮重要作用,為揭示周期節(jié)律破壞與乳腺癌發(fā)生發(fā)展間關(guān)系的分子機(jī)制提供了理論依據(jù)。
[Abstract]:The destruction of the periodic rhythm is closely related to the occurrence of a variety of hormone related tumors, such as the occurrence of breast cancer, and the abnormality of the estrogen receptor ER signaling pathway is an important inducer of the occurrence of breast cancer, but the molecular mechanism between the cycle rhythm and the development of the tumor is not clear. Core clock protein CLOCK (circadi An Locomoter activity kaput) is an alkaline helix loop helix (basic helix-loop-helix/Per-Arnt-Sim homology domain, bHLH/PAS) protein containing bHLH/PAS domain. In the periodic rhythm, CLOCK usually forms a heterogenous two polymer with BMAL1 to regulate the transcription of the downstream genes, which plays an important role in the maintenance of periodic rhythms. There is little research on whether CLOCK directly participates in tumor related pathways.
The post-translational modifications of the proteins include phosphorylation, acetylation, SUMO, and ubiquitination. These post-translational modifications play an important role in regulating the function of proteins. There are few studies on post-translational modification of CLOCK, and only it can be phosphorylated by PKC (protein kinase C) and GSK-3 (glycogen synthase kinase-3). One point is the study of the SUMO modification of CLOCK, and the effect of SUMO modification on its function, especially the effect on the estrogen signaling pathway. This study focuses on the MCF-7 cells positive for estrogen receptor ERa, and the main work is as follows:
1. the interaction between the clock protein CLOCK and the estrogen receptor ERa was found in the MCF-7 cells, and the interaction between the two was enhanced by estrogen (E2) stimulation. Through the reporter gene experiment, it was found that in the presence of estrogen, CLOCK has a significant promoting effect on the transcriptional activity of ERa, indicating that CLOCK may be the auxiliary activation of ERa. The factor is involved in the estrogen signaling pathway mediated by ERa.
2. through structural and sequence analysis, we found that CLOCK has 2 potential SUMO modification sites, that is K67 and K851, indicating that CLOCK may be a SUMO modified substrate. Then, CLOCK is proved to be SUMO in MCF-7 cells, while SUMO chemokine of CLOCK in the condition of E2 stimulation, 3 mutants are constructed by fixed-point mutation. CK K67R, K851R, K67R/K851R (2K/2R). Unit point mutation (K67R or K851R) decreased the SUMO level of CLOCK compared with the wild type, while the dual loci mutant 2K/2R made CLOCK SUMO disappear and reduced its own transcriptional activity. SUMO was removed and the transcriptional activity of CLOCK was reduced.
3. through the nuclear separation and immunofluorescence experiments, it was found that SUMO CLOCK was mainly distributed in the nucleus, and SUMO reduced the distribution of CLOCK in the nucleus. Further research found that the SUMO double site mutant of CLOCK, that is, CLOCK2K/2R, weakened the interaction between CLOCK and BMAL1, thus reducing the transcriptional activity of CLOCK and in the cell. The location in the nucleus.
4. SUMO modification usually changes the structure of the protein and affects the interaction between the substrate protein and other proteins. Through the immunoprecipitation experiment, it is found that the de SUMO CLOCK weakens the interaction between CLOCK and ER a. Then, the reporter gene experiment shows that SUMO CLOCK can enhance the ER alpha transcriptional activity because of the wild type CLOCK. Overexpression can significantly activate the transcriptional activity of ER alpha, whereas the SUMO mutant CLOCK, CLOCK2K/2R, has lost this function.
5. Cyclin D1 is the classic downstream target gene of ER alpha and promotes cell proliferation. Through real-time quantitative PCR and reporter gene experiments, it is found that wild type CLOCK can activate the expression of Cyclin D1, but CLOCK2K/2R can not. Meanwhile, the proliferation of cells is studied by MTT, and wild type CLOCK can promote MCF-7 cells. Proliferation, flow cytometry also concluded that the over expression of wild type CLOCK decreased the number of G0/G1 cells and increased the number of S cells, while the overexpression of the double mutant of CLOCK had little effect on the cell cycle.
Through this study, we linked the clock protein CLOCK with the estrogen signaling pathway mediated by ERa, and identified an important post translation modification and SUMO transformation of CLOCK. It is worth noting that the SUMO modification plays an important role in the regulation of CLOCK in the estrogen signaling pathway, in order to reveal the development of periodic rhythm damage and the development of breast cancer. The molecular mechanism of the inter relationship provides a theoretical basis.

【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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本文編號(hào)：1798494