【作者】 薛赓；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2010， 博士

【摘要】 作为实体瘤的主要特征之一,缺氧(Hypoxia)通常被精确定义为组织中的氧分压低于5～10毫米汞柱。众所周知,恶性细胞的生长同样需要血管网络为其提供充足的氧和养分,但是肿瘤细胞的恶性增殖总是远远快于血管网络的形成,当组织中的血管网络无法满足肿瘤细胞的生长、代谢的需要时,就形成了局部缺氧的微环境。缺氧微环境能够影响肿瘤细胞的很多生物学特性,其中包括细胞的生长、凋亡、新生血管的生成、转移以及对治疗的敏感性等等。因此深入探讨肿瘤缺氧微环境的形成机制以及肿瘤细胞在缺氧微环境中的应对机制就成为理解肿瘤发生、发展等生理过程的关键所在,而对肿瘤发生、发展过程更深刻的理解又能够促使人们找到肿瘤诊断和治疗的新途径。microRNAs (miRNAs)是一类长度约为22个核苷酸(nucleotide, nt)的非编码小分子RNA, miRNAs参与了包括细胞分化、增殖、代谢、死亡以及肿瘤生成等在内的一系列的细胞生理和病理进程。近年来大量的研究已经证实,miRNAs与肿瘤的缺氧微环境之间也存在着千丝万缕的联系。相比正常的组织,肿瘤组织中miRNAs的整体表达水平往往会发生巨大的改变,研究人员已经证实,在某些肿瘤中这种改变与肿瘤的临床、病理特征密切相关,但是直到今天,人们还未能对这种变化的意义做出确切的解释,miRNAs在肿瘤缺氧微环境中的调控和作用机制仍然有待于进一步的探索和发现。为了探讨miRNAs与实体瘤缺氧微环境的相关性及其参与肿瘤发生、发展调控的具体机制,借助于高通量筛查手段——miRNAs芯片,我们对培养于不同氧浓度下的结直肠癌细胞系中miRNAs的表达情况进行检测和鉴定,筛选出了多种在缺氧状态下表达水平发生改变的miRNAs。在此基础之上,进一步通过体外、内实验对其中一些重要的缺氧相关miRNAs的调控和作用机制进行了深入的探讨。我们的研究显示,缺氧状态下培养的p53野生型细胞中miR-17-92簇的表达水平显著下调,而同样在缺氧状态下培养的p53突变型细胞中则没有变化,且这种缺氧依赖性的表达下调并不受已明确的miR-17-92簇转录调控因子c-Myc的控制；通过报告基因检测系统,我们发现miR-17-92簇启动子区的近端存在一个p53结合位点,通过与该位点的结合,p53在缺氧状态下对miR-17-92簇的表达水平进行抑制;免疫共沉淀、重复免疫共沉淀以及定量分析的结果显示,该位点与基本转录元件——TATA框重叠,p53与基本转录因子——TATA结合蛋白能够竞争性的结合于启动子上的这一区域;上调miR-17-92簇的表达能够明显抑制缺氧介导的细胞凋亡,而下调miR-17-92簇中主要成员miR-17-5p和miR-20a的表达会促进缺氧介导的细胞凋亡;以上这些结果都强烈提示,p53依赖性的miR-17-92簇表达下调在缺氧介导的细胞凋亡中发挥着重要的作用,因此我们的发现促进了人们对于p53的肿瘤抑制功能的理解。与此同时,我们还发现肿瘤的缺氧微环境能够导致miR-15-16簇的表达下调,进而影响肿瘤细胞的增殖、凋亡尤其是迁移和侵袭能力;通过结直肠癌原位动物模型,我们发现较高的miR-15-16簇表达水平能够抑制肿瘤的新生血管生成和远端转移(主要是肝转移);进一步的体外、内实验显示,miR-15-16簇通过靶基因——成纤维细胞生长因子-2实现对抑制肿瘤的新生血管生成和远端转移的调控;临床相关性分析显示,miR-15-16簇的水平与结直肠癌患者的临床、病理特征密切相关,miR-15-16簇的表达水平能够作为潜在的标志物指示结直肠癌患者的预后；以上这些结果提示,肿瘤的缺氧微环境能够介导miR-15-16簇的表达下调,进而导致成纤维细胞生长因子-2失去转录后水平的抑制调控,从而达到促进肿瘤新生血管的生成以及肿瘤的远端转移的目的,我们的发现促进了人们对于肿瘤细胞在缺氧微环境中的应对机制的理解。综上所述,本研究为实体瘤缺氧微环境的深入理解以及实体瘤的早期诊断和靶向治疗提供了新的思路。更多还原

【Abstract】 Hypoxia remains a major characteristic of solid tumors and defined but usually referring to tissues at oxygen levels below 5 to 10 mmHg. Malignant cell growth requires the presence of a local vascular network that supplies oxygen and nutrients. However, a highly proliferating mass of tumor cells develops faster than the vasculature, and tumor cells rapidly meet up with an avascular environment deficient in oxygen. Hypoxia affects a variety of tumor cell properties such as cell growth, apoptosis, neovascularization, metastasis and sensitivity to treatment. Therefore, how hypoxic environments are generated in tumor tissues and how cells respond to hypoxia are essential questions in understanding tumor progression and a better understanding of these processes may lead to novel strategies for diagnosis and treatment. microRNAs (miRNAs) are about 22 nucleotide RNA molecules that participate in a wide variety of physiological and pathological cellular processes such as cell differentiation, proliferation, death, metabolism and more recently tumorigenesis. Recent studies have established a link between a distinct miRNAs expression profile with hypoxia, and global miRNAs expression changes have been described to occur in human cancers and in some cases shown to correlate with the clinicopathological features of the tumor. However, there is still few mechanism has been proposed to date for these profile alterations and relatively little is known about miRNAs regulation and response to hypoxia.In order to propose the mechanism of miRNAs response to hypoxia and involve in tumorigenesis or tumor progression, we screened the miRNA expression patterns in colorectal cancer (CRC) cell line cultured in diverse oxygen pressure using a high-resolution mapping assay, miRNA microarray and identified a specific group of hypoxia-related miRNAs with aberrant expression level. And then, the regulatory and acting mechanism of some important hypoxia-related miRNAs was investigated in vitro and in vivo. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc, a promised transcription regulator of miR-17-92 cluster. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53-and the TATA-binding protein (TBP)overlap within the promoter; these proteins were found to compete for binding. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumor suppressive function of p53. Furthermore, we also demonstrate that the levels of miR-15-16 cluster are remarkably decreased under hypoxia and the low-expression favors colorectal cancer cell apoptosis, proliferative and especially invasive/metastatic behavior. Intratumoral administration of miRNAs shows that high levels of miR-15-16 cluster can significantly present angiogenesis and metastasis in a CRC nude mouse model, fibroblast growth factor-2 (FGF2), which regulates tissue angiogenesis, was identified as the direct and functional target of miR-15-16 both in vitro and in vivo. We also correlated the expression of miR-15-16 cluster with distant metastasis and clinical outcomes in malignant tissues. These data indicated that down-regulation of miR-15-16 cluster expression induced by hypoxia in CRC promotes tumor angiogenesis and distant metastasis by enhancement of FGF2 expression. In a word, our study provides a better understanding on the specificity of hypoxia and thus may be helpful in early diagnosis and targeting therapy of solid tumors更多还原