乳腺癌细胞中CD74乙酰化调控的分子机制

发布时间：2018-05-25 06:10

本文选题：YB1 + CD74　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景癌症又称恶性肿瘤,是人类主要的致死疾病之一。作为女性中发病率最高的一类癌症,乳腺癌使女性的健康受到了严重的威胁。而该疾病最主要的致死因素就是癌细胞的转移扩散引起的多器官病变。因此,乳腺癌转移机制的研究对其治疗有着非常重要的意义。CD74在人类中共有四种亚型:p33,p41,p35,p43,我们在乳腺癌中主要检测到的是p35这一亚型。CD74可以发生多种翻译后修饰,如N/O糖基化,磷酸化,棕榈酰化;而CD74乙酰化这一修饰还未见报道。CD74最初被发现是作为MHCII的分子伴侣,在抗原递呈中起重要作用。随后发现CD74也具有不依赖MHCII的功能,如作为细胞表面受体,即巨噬细胞迁移抑制因子(MIF)的受体,引起信号级联反应;调控树突状细胞迁移等。我们的前期数据表明,CD74调控乳腺癌细胞转移,而乳腺癌细胞中CD74表达调控的分子机制并不清楚。YB1(Y-Box binding protein 1)在结构上分为多变非保守的N端结构域,高度保守的冷休克结构域和C端结构域三部分。N端结构域含有丰富的丙氨酸和脯氨酸,与转录激活有关;C端结构域由酸性残基和碱性残基交替组成,与调节蛋白与蛋白之间的相互作用有关。YB1最初被发现是作为转录因子结合到MHC II的启动子区。YB1在DNA、mRNA和蛋白质多种水平上发挥作用,如参与DNA的修复、前体mRNA的剪切、维持mRNA的稳定性以及蛋白与蛋白之间的相互作用等。前期工作中,我们利用软件分析,发现在CD74的启动子区以及CD74的mRNA上都可能存在YB1的调控位点。因此,我们将重点研究YB1是如何调控CD74从而影响细胞转移的,以期寻找到新的化疗靶标。研究方法1.免疫印迹检测YB1 siRNA干扰实验对CD74及细胞运动相关蛋白表达的影响2.RT-PCR实验检测YB7 siRNA干扰实验在CD74转录水平的影响3.免疫印迹检测YB1siRNA干扰实验对CD74蛋白稳定性的影响以及免疫共沉淀实验探究YB1siRNA干扰实验对CD74泛素化的影响4.免疫共沉淀实验探究CD74的乙酰化修饰以及质谱检测CD74可能发生乙酰化的氨基酸位点5.免疫共沉淀实验探究YB1对CD74乙酰化的影响6.免疫共沉淀实验探究YB1和CD74以及其他相关蛋白之间的相互作用7.免疫共沉淀实验探究SIRT1对CD74乙酰化的影响8.免疫共沉淀实验探究三位点突变的CD74与YB1、SIRT1、CD44之间结合能力的改变9.Transwell实验检测CD74乙酰化对细胞侵袭的影响10.免疫共沉淀实验探究CD74乙酰化与CD74泛素化之间的关系实验结果1.敲低YB1下调CD74和细胞运动相关蛋白的表达2.YB1调控CD74的稳定性是在蛋白水平上而非转录水平3.CD74存在乙酰化修饰,并且YB1影响CD74的乙酰化4.CD74与YB1、SIRT1和HDAC1蛋白之间存在相互作用5.SIRT1影响CD74的乙酰化水平6.质谱分析CD74潜在的乙酰化位点分别是Lys80、Lys99和Lys106,将赖氨酸突变为精氨酸后CD74的乙酰化水平减弱,泛素化水平降低7.CD74乙酰化位点突变影响CD74与YB1、SIRT1、CD44之间的结合8.CD74乙酰化位点突变影响细胞侵袭实验结论在人乳腺癌细胞中,我们发现YB1通过影响CD74与去乙酰化酶SIRT1之间的结合调控CD74的乙酰化修饰以及CD74的稳定性,CD74可能的乙酰化赖氨酸位点突变后,其乙酰化水平降低,泛素化水平减弱,与YB1和SIRT1的结合能力增强,与CD44的相互作用减弱。最终,CD74乙酰化位点突变使得细胞侵袭能力降低。
[Abstract]:Background cancer, also known as malignant tumor, is one of the major fatal diseases of human beings. As a type of cancer with the highest incidence in women, breast cancer is a serious threat to women's health. The most fatal factor in the disease is the multiple organ lesion caused by metastasis and diffusion of cancer cells. Therefore, the mechanism of breast cancer metastasis is studied. There are four types of.CD74 subtypes in human being: P33, p41, p35, p43. Our main detection in breast cancer is that p35 this subtype.CD74 can undergo a variety of post-translational modifications, such as N/O glycosylation, phosphorylation, palmiylation, and CD74 acetylation has not been reported that.CD74 was initially found to be done. The molecular chaperone for MHCII plays an important role in antigen presentation. Subsequently, it is found that CD74 also has functions that do not depend on MHCII, such as cell surface receptor, macrophage migration inhibitory factor (MIF) receptor, signal cascade reaction, and regulation of dendritic cell migration. Our preliminary data show that CD74 regulates breast cancer cell metastasis, The molecular mechanism of the regulation of CD74 expression in breast cancer cells is not clear that.YB1 (Y-Box binding protein 1) is structurally divided into a variable non conservative N terminal domain. The highly conserved cold shock domain and the three.N terminal domain of the C end domain are rich in alanine and prolysine, which are related to the activation of the transcriptional activity; the C terminal domain is an acid residue. Alternately composed of base and alkaline residues, related to the interaction between protein and protein,.YB1 was initially found to play a role in DNA, mRNA, and protein levels in the promoter region of MHC II as a transcription factor, such as the repair of DNA, the shear of the precursor mRNA, the stability of the mRNA and the protein and protein. In previous work, we use software analysis to find that there may be YB1 regulatory sites in the promoter region of CD74 and on the mRNA of CD74. Therefore, we will focus on how YB1 regulates CD74 and thus affects cell metastasis in order to find new chemotherapeutic targets. Method 1. Western blot to detect YB1 siRNA dry. Effects of disturbing experiments on the expression of CD74 and cell related proteins in 2.RT-PCR test the effect of YB7 siRNA interference test on the transcription level of CD74 3. the effect of YB1siRNA interference test on the stability of CD74 protein and the effect of immunoprecipitation experiment on the effect of YB1siRNA interference experiment on CD74 ubiquitination, 4. immunoprecipitation experiment Exploring the acetylation modification of CD74 and mass spectrometric detection of CD74 possible acetylation of amino acid site 5. immunoprecipitation experiments to explore the effect of YB1 on CD74 acetylation 6. immunoprecipitation experiments explore the interaction between YB1 and CD74 and other related proteins 7. immunoprecipitation experiments explore the effect of SIRT1 on CD74 acetylation 8. immunity A co precipitation experiment to explore the effect of the binding ability of CD74, YB1, SIRT1, CD44 between three loci mutations in the 9.Transwell test the effect of CD74 acetylation on the invasion of cells 10. the experimental results of the relationship between CD74 acetylation and CD74 ubiquitination: 1. knock down the low YB1 downregulation CD74 and the expression of the expression of cell motion related proteins The stability of control CD74 is at the protein level and non transcriptional level 3.CD74 acetylation modification, and YB1 affects the acetylation of CD74, 4.CD74 and YB1, SIRT1 and HDAC1 protein interaction between 5.SIRT1 and CD74, the acetylation level 6. mass spectrometry analysis CD74 potential acetylation sites are Lys80, mutation and lysine mutation The level of acetylation of CD74 after arginine decreased, and the level of ubiquitination reduced 7.CD74 acetylation site mutations affecting CD74 and YB1, SIRT1, CD44 binding 8.CD74 acetylation site mutation affecting cell invasion experiment conclusion in human breast cancer cells, we found that YB1 regulates CD74 by the binding between CD74 and deacetylase SIRT1. Acetylated modification and the stability of CD74, after the mutation of CD74, the level of acetylated lysine was reduced, the level of acetylation was reduced, the level of ubiquitination was weakened, the binding ability with YB1 and SIRT1 increased, and the interaction with CD44 weakened. Finally, the mutation of CD74 acetylation site reduced the cell invasiveness.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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