铜离子相关基因CUTA的功能初探暨RLIM对肝癌的抑制作用研究

【作者】 杨景春；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 铜离子是人体所必需的微量元素,在体内发挥重要作用,但是过量铜离子会产生大量反应性氧自由基离子(ROS),造成细胞的氧化性损伤,导致人类各种疾病的发生,肝豆状核变性(Wilson disease)是常见的铜离子代谢缺陷性疾病。但近年来的研究发现铜离子毒性还与阿尔茨海默氏症,动脉粥样硬化等的发生紧密相关。许多与疾病直接相关的蛋白如微管相关蛋白Tau,淀粉样前体蛋白APP,软病毒蛋白Prion都是铜离子结合蛋白,这些蛋白与铜离子的相互作用是导致疾病发生的重要因素之一。因此研究潜在的铜离子相关基因及其功能,将有助于揭示铜在人类疾病中的作用机制。人类的CUTA(copper tolerance protein A又名cation tolerance protein A)蛋白是CutA1同源蛋白中的一员,此类同源蛋白多数具有铜离子结合能力,并且大肠杆菌中的CutA1蛋白可能参与了细菌对铜的容忍性。CUTA蛋白与大肠杆菌CutA1蛋白的同源性提示我们CUTA的功能可能与铜离子平衡相关,本文对人类CUTA基因的表达,蛋白性质,以及与铜离子的相关性等方面进行了初步的探讨。我们发现CUTA在人类的18种组织中广泛表达,在肝脏和前列腺中有较高水平的表达。CUTA蛋白集中分布在细胞核的周围,大部分能与线粒体染料共定位,是一种潜在的线粒体蛋白,DSG(disuccinimidvlglutarate)交联试验显示,CUTA蛋白以三聚体的形式存在于真核细胞中。随后,我们对CUTA蛋白与铜离子的相关性进行了深入的探讨,发现CUTA蛋白与铜离子的代谢平衡紧密相关,首先,CUTA蛋白能够结合多种二价金属离子,但它对二价铜离子的结合能力最强;其次,CUTA的表达受铜离子浓度的影响,CUTA蛋白的表达量随细胞外铜离子浓度的增高而增多;最后,CUTA超量表达时,影响了细胞对铜离子毒性的敏感性,使细胞对高浓度铜离子的毒性变得更为敏感,进一步的分析表明CUTA促进了铜离子对细胞增殖的抑制作用,增强了铜离子对细胞凋亡的诱导作用,从而使细胞对铜离子毒性更为敏感。在研究过程中,我们还通过免疫新西兰大白兔的方法制备纯化了CUTA特异性的多克隆抗体,并用此抗体检测了CUTA蛋白在癌症细胞株及肝癌和癌旁组织中的表达。发现CUTA蛋白的表达量与肝癌的发生无明显的相关性,但是在不同的肝癌样本中,CUTA蛋白有不同的上下调表达,这提示我们CUTA可作为肝癌分子分型的候选基因。此外,本文还对本实验室筛选到的一个肝癌中显著下调表达的基因-泛素化连接酶RLIM,在肝癌发生发展中的作用展开了研究,我们依次检验了RLIM的表达对肝癌细胞的克隆形成率,增殖速率和移植瘤生长速度的影响。结果表明:1.RLIM的表达显著降低了肝癌细胞的独立生存能力,抑制了肝癌细胞的克隆形成率,RLIM转染的SK-Hep1细胞的克隆形成率只有对照组克隆形成率的25%;2.RLIM的表达显著抑制了肝癌细胞的增殖速度,比较RLIM稳定转染的SK-Hep1细胞株和对照细胞株的增殖曲线,发现RLIM稳定转染的SK-Hep1细胞的增殖速度显著慢于对照细胞的增殖速度。3.RLIM的表达抑制了肝癌细胞移植瘤的生长速度,将RLIM稳定转染的细胞和对照细胞分别接种于裸鼠,检测肝癌细胞移植瘤的生长速度,发现RLIM稳定转染的细胞株的移植瘤,其生长速度显著慢于对照组细胞的移植瘤,并且最终形成的肿瘤的大小和重量均小于对照组。这些结果暗示了RLIM是一个新的潜在的肝癌抑制基因。综上所述,本文通过对CUTA的组织表达谱,蛋白的亚细胞定位和多聚体构象的分析检测,发现并初步证明了CUTA是一个广谱表达的线粒体蛋白,具有三聚体构象,在对CUTA的功能研究中,我们发现CUTA的功能与铜离子紧密相关,CUTA蛋白能够结合铜离子,CUTA的表达受铜离子浓度的影响,CUTA超量表达能够影响细胞对铜离子毒性的敏感性,因此,我们推测CUTA是人体内的一个铜离子相关蛋白,其功能可能与铜离子对细胞的毒性作用有关。此外,对泛素化连接酶RLIM在肝癌发生发展中的作用的研究显示,RLIM的表达不仅对肝癌细胞的克隆形成和增殖速度具有抑制作用,而且对肝癌细胞移植瘤的生长具有抑制作用,因此我们推测RLIM是一个潜在的肝癌抑制基因。更多还原

【Abstract】 Copper,an essential trace element,plays an important role in biological system.However, excessive copper is toxic and contributes to production of excessive reactive oxygen species (ROS),which results in the oxidant damages in the cell and genesis of human diseases. Mounting evidence is showing the relativity of copper with many human diseases,such as Alzheimer’s disease,atherosclerosis,Creutzfeldt-Jakob(CDJ) disease and so on.Those proteins directly involved in the diseases genesis,such as the microtube related protein Tau, amyloid precursor protein APP and Prion proteins,can bind copper,and the interaction between those proteins and copper plays an important role in diseases.So study on the potential proteins associated with copper will be helpful to reveal the role copper plays in diseases.Human CUTA(copper tolerance A also named as cation tolerance protein A) is a homologous gene of CutA1 in Escherichia coli.Most CutA1 homologous protein have the capability of copper binding,and the CutA1 protein in Escherichia coli is involved in copper tolerance.So the sequence characterization of CUTA protein indicated that CUTA is a potential protein associated with copper.However,the function of CUTA gene is still unknown.In order to reveal the potential function of CUTA gene,we made a primary study on the tissue expression pattern of CUTA gene,the characterization of CUTA protein,and its relativity with copper.It was found that CUTA was constitutively expressed in human tissues and expressed with a high level in liver and prostate.The results also showed that CUTA protein concentrated in the perinuclear region,where it co-localized in a large part,but not completely,with mitochondria. CUTA is a potential mitochondrial protein with the structure of trimers proved by cross-linking with DSG(disuccinimidyl glutarate).In the study on the association of CUTA protein with copper,we found that CUTA protein has the strongest binding ability with Cu(Ⅱ) in the detected metals.In addition,the expression of CUTA protein was increased when the cells were exposed to high concentration of copper.Furthermore,CUTA could affect the sensitivity of the cells to the copper toxicity.Over-expression of CUTA seemed to sensitize the cells to copper toxicity.All of the studies indicated the strong association between CUTA and copper.In the study,we raised and purified CUTA specific antibody by immunizing the New Zealand rabbit.The native CUTA protein could be detected in the different cancer cell lines.The expression of CUTA protein detected in the 21 pares of liver cancer samples showed that there was no significant association between CUTA and liver cancer.However, CUTA was significantly up or down regulated in some.liver cancer samples.So it seemed that CUTA could be used as a candidate gene for typing the liver cancer.In addition,in an effort to identify HCC relevant genes,we used differential display and found RLIM(RING finger LIM domain-binding protein) was significantly down-regulated in HCC.RLIM is an E3 ubiquitin ligase with a RING finger.To investigate the role RLIM plays in HCC,we checked the effect of RLIM gene expression on colony formation,cell. proliferation and tumor growth.The results showed that:1.RLIM over expression in SK-Hepl significantly decreased the efficiency of colony formation by 75%.2.RLIM expression in SK-Hepl(stably tansfeeted with RLIM) significantly inhibited the cell proliferation.3.RLIM over-expression inhibited the tumor growth.This was done by replanting the stably transfeeted cells in nude mice,and then measuring the growth rate,size and weight of the tumor originated from the cancer cells.In general,we found and primarily proved that CUTA is a mitochondrial protein widely expressed in tissues,forms trimer in cell lysate.We also found that CUTA could bind copper and change the sensitivity of the cells to the copper toxicity,and the expression of CUTA was affected by copper concentration.So we considered the function of CUTA was associated with copper and CUTA maybe play a role in copper toxicity.We constructed two powerful tools for the research in CUTA gene.One is the CUTA stably transfected HeLa cells and the other is the CUTA specific antibody.In addition,we investigated the role of RLIM in HCC because RLIM was found to be significantly down-regulated in HCC.We found that over-expression of RLIM in SK-Hepl cells not only inhibited the colony formation and cell proliferation,but also inhibited the growth of tumor in nude mice.So it was considered that RLIM is a new potential tumor suppressor gene in HCC.更多还原