斑马鱼p53蛋白与其自身mRNA相互作用的研究

【作者】 赵向忠；

【导师】 林秀坤；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋药物学， 2010， 博士

【摘要】 p53基因是迄今发现的与人类肿瘤相关性最高的肿瘤抑制基因,50%以上人类肿瘤疾病的发生与p53基因突变或功能失调有关。由p53基因编码的p53蛋白作为一种重要的转录调控因子参与细胞周期控制、细胞增殖与分化、细胞凋亡、发育等重要生命活动。正常情况下,细胞内p53通过泛素依赖的降解途径而维持在较低水平。在各种应激条件如DNA损伤、缺氧及营养缺乏等情况下,p53稳定性增强、富集并被激活、作为基因特异性调控因子调控与细胞周期控制、凋亡诱导、DNA损伤修复等相关的一系列靶基因的表达。p53蛋白本身的表达、稳定性及活性控制在细胞内受到多水平精细调控,包括转录水平、翻译水平及翻译后水平调控等。其中翻译水平调控是细胞面对应激状态的一种重要调节方式。前期的研究已表明,人和鼠的p53蛋白可以与其自身mRNA的5’UTR或3’UTR特异性结合增强或抑制其自身mRNA的翻译,存在翻译水平自调控现象。近年来更有研究发现,除自调控外,多种蛋白质因子可调控p53 mRNA的翻译,如HuR和RPL26蛋白可与p53 mRNA结合,增强其在应激刺激下的翻译水平,快速增加p53蛋白的表达量,从而保护细胞或有机体免受损伤。而在正常状态下,核仁素等蛋白通过与p53 mRNA结合可抑制p53 mRNA的翻译,从而维持p53蛋白的低水平表达。作为模式生物和抗肿瘤药物筛选模型的斑马鱼,其p53信号通路与人类p53信号通路调控机制极其一致,因此斑马鱼作为p53信号通路的高通量筛选模型,在其体内是否也存在p53蛋白与其自身mRNA的相互作用,这种相互作用对斑马鱼发育是否有影响及在应激条件下有何重要作用意义尚未见相关报道。本论文在此研究背景下,首先利用原核表达系统克隆表达了斑马鱼p53蛋白,通过体外紫外交联及免疫共沉淀(IP)：RT-PCR技术证实重组表达的p53蛋白在体外能与其自身mRNA特异性结合。为进一步验证斑马鱼p53蛋白在体内是否存在自调控作用,我们构建了p53 mRNA的荧光素酶报告载体和斑马鱼p53蛋白真核表达载体,通过共转染p53 mRNA的荧光素酶报告载体和斑马鱼p53蛋白真核表达载体,在细胞水平,通过荧光素酶报告系统检测荧光素酶活性,证明在细胞内p53蛋白能与其身mRNA结合,并且在喜树碱(CPT)、UV等DNA损伤应激条件下,表现为荧光素活性增强,表明这种结合在应激条件下是增强翻译的。将上述质粒显微注射斑马鱼胚胎进行斑马鱼在体实验亦进一步证实二者在体内的相互作用,这种相互作用在应激处理时可增强p53的翻译表达,提示这种相互作用可能在应激状况下对斑马鱼正常发育起保护作用。我们的研究结果表明斑马鱼p53蛋白与其自身mRNA之间存在相互作用,并且这种相互作用对斑马鱼发育有重要影响。并且进一步阐明了斑马鱼的相应生物学过程与人的相似性,该研究将为从p53调控途径中寻找癌症治疗的新靶标提供理论基础和重要线索,同时为斑马鱼筛选平台的构建和进一步推广应用奠定基础。更多还原

【Abstract】 The tumor suppressor p53 gene, which is an important factor involved in the occurrence and development of cancer, plays a key role in regulating the cell cycle, cell growth, and development. More than half of human malignancies are due to a mutation or functional disorder of the p53 gene. Under normal conditions, the expression of p53 protein is maintained at low levels through the ubiquitin degradation pathway. In response to various cellular stresses, such as DNA damage, hypoxia, or nutrient deprivation, the tumor suppressor gene produces p53 protein as a sequence-specific transcription factor to regulate expression of a series of target genes involved in governing cell cycle arrest, apoptosis, and DNA repair. The expression of p53 protein is regulated at different levels, including transcriptional regulation, translational regulation, and post-translational regulation.Translational regulation of p53 is an important mechanism involved in the response to various stresses. Studies have shown that p53 protein can bind with the 5’UTR or 3’UTR region localized at its cognate mRNA and repress its translation. The UTRs of both human and murine p53 are predicted to form stable stem-loop structures that are capable of inhibiting translation. Moreover, some protein factors may interact with the 5’UTR or 3’UTR domain of p53 mRNA, resulting in enhancement or decreasing in p53 translation. HuR and RPL26 protein have been shown to bind with the 3’UTR of p53 mRNA and regulate it by enhancing translation or increasing its stability. This type of regulation can protect cells or organisms against damage or abnormality.p53 protein is an important regulation factor that can bind to p53 mRNA to regulate its translation in human and murine. To determine if a similar interaction exists in zebrafish and if the interaction affects zebrafish development, we cloned and expressed p53 protein from zebrafish in Escherichia coli. The binding activity of p53 protein and its mRNA was determined by UVcross-linking and immunoprecipitation: RT-PCR analysis. And then the p53mRNA was cloned into the psiCHECK-2 vector, between the XhoI and Not I sites, immediatedly 3’downstream from the Renilla luciferase gene. The constructed luciferase reportor and eukaryotic expression vector for p53 were cotransfected into cells or were microinjected into zebrafish embryos. The luciferase activities were measured with Dual luciferase reporter system assay. Our results demonstrated that p53 protein could enhance luciferase translation by binding to its mRNA. Further experiments were carried out, immunoprecipitation: RT-PCR, microinjection of zebrafish showed that 53 protein could specifically bind to its own mRNA in zebrafish embryos. This interation play important role in zebrafish development.Our result demonstrated that p53 protein could specifically bind to its own mRNA in zebrafish embryos. p53 protein could enhance mRNA translation in cells and in zerafish embryos under stresses. This interatction may be an important protective mechanism for zebrafish development facing stresses. Our result also provided evidences that zebrafish may be developed as useful model for studying the anti-tumor agents targeting p53 signal pathway.更多还原