【作者】 陈亮；

【导师】 单革；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 p53蛋白,也被认为是肿瘤的抑制蛋白。自从发现以来,被证实参与细胞周期调控,激活DNA修复、维持基因组稳定及促进细胞凋亡。在超过50%的人类的癌症中都出现p53功能缺失,其中就包括单位点和多位点的突变及结构的扭曲。同样也有越来越多的研究证实,p53蛋白的突变,使得其空间构象发生变化,从而失去对细胞周期调控与促进细胞凋亡的重要生物学功能,与此同时,还会得到新的功能(Gain-of-Function, GOF),如促进细胞转移、侵染等,由抑癌基因变为癌基因。目前越来越多的研究组开始关注p53蛋白突变体的研究,其中就包括促进突变体的回复突变及抑制其生物学功能。此项研究对于癌症的靶点治疗具有重要的生物学研究与医学价值。核酸适配体(aptamer)是一段短的单链核酸分子,其特点是能够根据结构特异性针对性地地结合多种靶标分子,如蛋白质、小分子、糖、脂肪等。它是由一个叫SELEX (Systematic Evolution of Ligand Exponential enrichment)的体外筛选进程得到的。SELEX技术自从1990年由三个研究组同时发明并已广泛地运用到诸如核糖体开关、靶向治疗等生物及医学应用。于是我们有理由相信,通过此筛选方法,我们也可以筛选到对诸如p53突变体有高亲和力和特异性的核酸适配体。目前,还未见研究组筛选到p53突变体适配体的报导。在本论文中,我们的首要研究目的就是能够筛选到p53R175H的适配体。由于此突变型与野生型的唯一区别只在于第175位的氨基酸突变,所以我们对传统的SELEX筛选过程进行了改进,名为差别竞争SELEX筛选,将p53野生型与突变型蛋白质分别经过固相偶联,同时投入体系中,竞争性地结合库分子,最大限度地增加筛选特异性与差异性。经过5轮的差别竞争SELEX筛选,我们得到了21个p53R175H蛋白的候选适配体。通过亲和力检测,适配体p53R175H-APT与p53R175H蛋白的亲和力大大超过对于野生型p53的亲和力,并且在凝胶迁移实验中我们最终确定p53R175H-APT能够在体外和p53R175结合。所以我们最终确定的p53R175H-APT作为我们后续研究的对象。尽管在亲和力检测中,p53R175H-APT对于p53突变体有着相对于野生型p53较强的亲和力,但其细胞学效应还有待研究。在本论文中,我们试图阐述筛选到的适配体P53R175H-APT在细胞中的功能。首先,我们必须确定P53R175H-APT在其靶标的选择上具有严格的特异性。在含有p53野生型的HEK293T和HeLa细胞中,通过将p53R175H-APT转染入上述两种细胞中,无论从表型还是从细胞生长于凋亡的情况看,均没有发现与对照组有显著性差异。同时,在只含有p53的另一个突变体p53R273H的H1299细胞中,经过转染p53R175H-APT,相对于对照组我们依然没有观察到明显的差别。在完成了以上实验证实p53R175H-APT对于含有p53野生型与p53其他突变体的细胞没有显著性作用之后,我们将此适配体构建到质粒并转染入H1299-p53R175H稳转细胞系中,我们发现,经过p53R175H-APT处理的细胞,细胞增殖得到明显的抑制,细胞的死亡明显增多。而对于含有p53野生型的细胞,则没有此现象。侧面说明此RNA适配体可以特异性地识别p53R175H并显著影响细胞增殖与细胞凋亡。为了更加确定产生如此明显的生物学差别与p53突变体相关,我们通过p53的免疫沉淀证实了在细胞中,p53R175H-APT与p53突变体结合的确大于IgG和scramble序列对p53突变体的结合能力。与此同时,通过克隆形成实验与soft agar实验我们证明了通过此适配体处理的细胞,其恶性程度与生长能力也有明显降低。由于很多p53的突变属于功能获得型突变,肿瘤细胞的迁移能力会不同程度地提高。为此我们设计了Transwell迁移实验和wound healing实验,证实经过此适配体处理过的细胞其迁移能力得到明显降低。进一步,在分子水平上,p53R175H-APT处理的细胞表现出更像p53野生型的调控方式,多个p53相互作用基因均有激活,一定程度上说明此适配体可使p53突变体的功能回复。同时,我们通过免疫荧光更加直观地证明了,经过p53R175H-APT处理过的p53突变体,能够从构象上使其回复成p53野生型。为了从更高水平验证此效应可以运用到实体瘤的治疗中,我们建立了裸鼠肿瘤模型,并将适配体通过实体瘤注射与静脉注射两种方法进行治疗。相对于对照组,适配体组的裸鼠肿瘤明显减小并维持较低水平,甚至消失。通过对实体瘤切片的TUNEL凋亡检测染色,我们发现在适配体治疗组中的凋亡细胞明显多于对照组。更加证实了p53R175H-APT可以通过p53R175H作用于肿瘤细胞,促进肿瘤细胞的凋亡、降低细胞生长能力、限制肿瘤细胞的迁移及具有一定程度的临床潜力。综上所述,本论文的研究成果阐述了一个新的SELEX筛选方法,并通过此方法成功筛选出与野生型p53只有一个碱基差异的p53R175H的适配体p53R175H-APT。体外实验证实其可以抑制肿瘤细胞生长并促进其凋亡,体内实验进一步证实了其潜在的临床价值,对于肿瘤靶向治疗提供新的思路。更多还原

【Abstract】 p53protein, also known as tumor suppressive protein, is involved in cell cycle regulation, activation of DNA repairing, maintaining genome stability and promoting cell apoptosis. p53loss of function is found in almost50%of the human cancers, including single site mutation, multisite mutagenesis and structural distortion. An increasing number of evidence shows that once p53is mutated, its conformation is alterd, resulting in lots of critical biological functions such as dysfunction of cell cycle regulation and promotion of cell apoptosis. Meanwhile, p53mutation also leads to an effect of Gain-of-Function (GOF) in cells like enhanced cell migration and invasiveness, changing from tumor inhibitor to tumor protein. Up to date, more and more research groups are focusing the p53mutants, from which reactivation of p53mutants and inhibition of their Gain-of-Function effects are the major points of the research, which makes it biologically and pharmacologically meaningful for the cancer targeted therapy.Aptamers are short single stranded nucleic acid molecules which can bind a variety of target molecules in a structure-dependent manner. Its targets can be proteins, small molecules, sugar, lipids etc.. It is screened out by a evolutionary procedure called SELEX (Systematic Evolution of Ligand Exponential enrichment). SELEX techonology was invented by three different research groups in1990and is widely used in many biological and pharmocological applications like riboswitch, targeted therapy. Thus we are confident that through this method we could screened out an aptamer that habors high affinity and specificity to the p53mutants.Up to date, there is no report about isolation of any p53mutant aptamers with high affinity. In this work, our primary goal is to screen out p53R175H aptamer. Since there is only one amino acid difference compared with its wild type, we then modified the conventional SELEX procedures as what we call Contrast Screening in which p53wild type and mutant were respectively coupled to different solid phase and were subject to competitive binding of their potential targets. This competitive system enhances, at a very high degree, the specificity of screening and the differences of the two proteins. After five rounds of selection, we obtained21aptamers that were believed to have much higher affinity towards p53R175H. Of them, p53R175H-APT was found to much higher affinity towards p53mutant over p53wildtype. In gel shift assay, we finally confirmed that p53R175H-APT and p53R175H have direct binding in vitro. We then set out to investigate the following functional study of p53R175H-APTAlthough p53R175H-APT exhibited higher affinity to p53R175H, its function in cell culture is yet to be investigated. In this work, we tried to illustrate how p53R175H-APT functioned in cells. Firstly we have to make sure that this kind of p53mutant aptamer has stringent specificity towards its targets rather than other molecules. We transfected p53R175H-APT to HEK293T and HeLa cells which habor p53wild type. No matter from phenotype nor cell growth and cell death, no significant difference was observed compared with sramble control. At the same time, in p53R273H expressing H1299cells, we also got no significant difference. After completion of the above experimets and making sure that p53R175H-APT has no significant function towards neither p53wild type and other p53mutant, We then established p53R175H-HI299stable cell line for our following experiments. We found that cell growth is significantly suppressed and cell death is increased after treatment of p53R175H-APT, which means p53R175H-APT specifically binds to p53R175H, inhibits cell growth and induce cell death. In order to make sure that so significant difference is due to the specific binding of p53R175H-APT and p53R175H, we carried out p53RIP assay and confirmed that in cell culture p53R175H-APT binds more to the p53mutant rather than IgG or scramble control. Meanwhile, clonogenic assay and soft agar assay indicated decreased colony formation ability. Since many p53mutants are GOF mutation, the ability of migration and invasiveness is enhanced after mutation, transwell assay and wound healing assay demonstrated that after treatment of p53R175H-APT, the migration and invasiveness are significantly decreased. Additionally in molecular level, p53R175H-APT treated cells could transactivate the p53target genes, which means rescue of p53mutant. In order to make it more direct, we performed immunostaining with p53wild type antibody PAbl620. The results indicated that structually p53R175H-APT treated p53mutant is more like with p53wild type which means a successful rescue of p53mutant.In order to verify the amazing effects of p53R175H-APT at a higher level in vivo, we established a nude mice xenograft model. Through intradermal injection and intravenous injection, we both found significantly smaller tumors in p53R175H-APT treated mice compared with scramble control. In TUNEL apoptosis staining, we found much more apoptotic cells in p53R175H-APT group, which indicated that p53R175H-APT functioned in vivo by inducing apoptosis of the cells. Above all, all the results leads to the conclusion that p53R175H-APT can promote tumor cell apoptosis, decrease the cell growth and clonogenesis, inhibit cell migration and transactivate p53target genes in cell culture and in vivo.Above all, this work illustrated a novel SELEX screening strategy by which we could successfully screen out an aptamer p53R175H-APT towards p53R175H, which only has one amino acid difference. In vitro experiments demonstrated that it could inhibit tumor cell growth and induce cell death, in vivo experiments further verified its potential clinical value. It offers a new insight to the cancer targeted therapy.更多还原