DEC1通过稳定cyclin E调节乳腺癌细胞增殖

发布时间：2018-06-22 12:46

本文选题：DEC1 + cyclin　；参考：《大连理工大学》2015年博士论文

【摘要】：全球每年都有数百万人死于癌症,癌症亦称恶性肿瘤,它的无限制、无止境地增生往往与细胞周期紊乱有关。越来越多的数据表明细胞周期蛋白cyclin E过表达或其基因扩增与肿瘤形成密切相关。cyclin E的表达受多种因子的调节,这些因子通过调节cyclinE的转录、翻译及翻译后修饰进而影响肿瘤的发生发展。时钟蛋白DEC1作为一个重要的转录因子,可以调节多种靶基因的表达,参与细胞分化、凋亡、衰老等生物学过程进而调控肿瘤的发生发展。目前的研究结果表明DEC1既可以抑制肿瘤的生长、介导p53诱导的细胞衰老,又可以促进肿瘤细胞的生存起到抗凋亡的作用,但是DEC1如何调节肿瘤细胞生存以及细胞周期进程的分子机理还不清楚。因此本文着重研究了DEC1如何影响乳腺癌细胞的增殖以及对细胞周期蛋白cyclin E稳定性及功能的影响。本文主要工作如下：1.通过克隆形成实验和MTT实验发现DEC1的过表达抑制了MCF-7和T47D细胞的生长。相反,干扰DEC1后明显抑制了细胞集落的大小和数量。2.在MCF-7细胞中通过免疫印迹、流式细胞术等方法研究DEC1表达的变化。实验发现时钟蛋白DEC1在细胞周期的进程中其自身表达存在周期性,主要在G1/S期表达量较高,其表达的变化与细胞周期蛋白cyclin E相似。在MCF-7和T47D两种乳腺癌细胞中,DEC1以剂量依赖的方式上调cyclin E蛋白含量,并且不影响cyclin E的mRNA水平。3.DEC1上调cyclin E的蛋白含量但不影响其转录,因此可能影响cyclin E的降解。通过半衰期实验证实DEC1稳定了cyclin E,延长了cyclin E的半衰期。通过免疫印迹实验,发现DEC1对cyclin E的调节依赖cyclin E的泛素E3酶Fbw7,通过免疫共沉淀实验证实DEC1抑制cyclin E与Fbw7a相互作用,抑制cyclin E的泛素化,进而稳定cyclinE。4.通过免疫共沉淀实验,哺乳动物双杂交实验证实在血清饥饿及正常条件下DEC1与周期蛋白cyclin E存在相互作用。同时,内源的实验也验证了DEC1与cyclin E存在相互作用。通过荧光共定位实验发现,cyclin E与DEC1很好的定位在细胞核中。另外,检测了DEC1与cyclin E相互作用动力学,结果发现DEC1与cyclin E主要是在G1及S期存在相互作用,在恢复培养8h后其相互作用最明显,即G1/S期检测点。5.研究DEC1对cyclin E功能及细胞周期进程的影响。cyclin E通常与Cdk2相互作用之后被降解,实现cyclin A与Cdk2的结合。通过共沉淀结果发现DEC1增加了cyclinE/Cdk2,抑制了随后的cyclin A/Cdk2的结合,引起细胞S期延长,导致细胞周期停滞。最后在裸鼠肿瘤实验中发现DEC1明显抑制了肿瘤的生长。本论文的研究将时钟蛋白DEC1与细胞周期因子cyclin E联系起来,确定DEC1抑制cyclin E泛素化降解过程影响了cyclin E的功能,进而抑制乳腺癌细胞的增殖,本研究为乳腺癌的治疗,特别是cyclin E高表达的乳腺癌的治疗提供理论依据,为选择特异性药物靶点、优化治疗措施提供科学基础。
[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.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

【相似文献】