【作者】 周卉卉；

【导师】 来茂德；

【作者基本信息】 浙江大学 ， 病理与病理生理学， 2010， 博士

【摘要】 结直肠癌是一类严重危害人类健康的恶性肿瘤。近年来,随着化疗、放疗和生物靶向治疗等多种手段的开展,死亡率有所下降,但发病率仍然很高。在中国,大肠癌发病率增长速度迅猛,尤其是苏浙沪三地。检出率低是影响大肠癌早期发现及治疗效果的重要因素之一。因此,因此积极开展结直肠癌发生机制的研究对于结直肠癌的防治具有很重要的现实意义。IGFBP-rP1 (insulin-like growth factor binding protein-related protein 1)是我们实验室在1999年用抑制性差减杂交法(suppresion subtractive hybridisation, SSH)构建的结直肠腺癌相对正常粘膜(T-N)文库中筛频率较高、在腺癌中高表达的一个基因。IGFBP-rP1又称为IGFBP7 (Insulin-like growth factor binding protein 7)、mac25 (meningioma associated cDNA 25)、PSF (prostacyclin-stimulating factor), AGM (angiomodulin)和TAF (tumor-derived adhesion factor).目前认为IGFBP-rP1与多种人类肿瘤关系密切,大部分报道认为IGFBP-rP1在多种人类肿瘤,如脑膜瘤、肺癌、肝癌等中均存在表达下调的现象,在肿瘤的发生发展中扮演抑癌基因的角色。基因的表达调控主要通过遗传学(genetics)和表遗传学(epigenetics)两种机制实现。遗传学改变是涉及核苷酸序列改变的机制,但是目前只能解释肿瘤发生机制中的一部分原因,表遗传学机制是一种不涉及DNA序列变化的可遗传的基因表达方式的改变,目前认为表遗传学是较遗传学更为常见的基因表达调控机制,许多具有重要功能的基因是通过表遗传学机制参与肿瘤的发生、发展。DNA甲基化表遗传的主要方式,是研究得最深入的作用方式,与抑制基因的表达有关,其在肿瘤发生中的作用是近年来的研究热点。实验室前期对IGFBP-rP1的表达调控进行了系列探索性工作,结果发现,IGFBP-rP1基因的启动子区域和所有5个外显子不存在序列突变,遗传学改变可能不是其主要转录调控机制。用亚硫酸氢钠-测序法(bisulfite sequencing PCR,BSP)详细研究基因5’端CpG岛中所有CpG位点的甲基化情况,在体内体外试验中均发现结直肠癌中,IGFBP-rP1基因的5’端CpG岛存在异常甲基化,并且第一外显子的异常甲基化水平与该基因的表达水平呈负相关。研究表明,DNA甲基化主要通过DNA甲基转移酶(DNA methyltransferases, DNMTs)的催化下,利用S-腺苷甲硫氨酸提供的甲基,将胞嘧啶的第5位碳原子甲基化,从而使胞嘧啶转化为5-甲基胞嘧啶。在人类,DNMTs主要有三种：DNMT1、DNMT3a与DNMT3b。DNMT3a和DNMT3b主要是使未甲基化的位点发生甲基化(从头发生甲基化,de novo methylation)。DNMT1则维持相关位点甲基化,并将甲基化信息传给子代细胞(维持甲基化,maintenance methylation)。5’-Azadc等去甲基化药物可能通过抑制DNMTs的活性实现去甲基化。EZH2 (enhancers of zest homologue 2)在肿瘤DNA甲基化中的作用最近受到关注。EZH2是一种转录抑制因子,也是一个组蛋白甲基化酶(histone methyltransferases, HMTs),属于Polycomb group (PcG)家族成员,是Polycomb repress complex 2/3 (PRC2/3)的重要成分。含EZH2的PRC复合物与H3K27包装成一个特别的染色质结构,该结构可能通过募集DNMTs,调控基因发生甲基化。IGFBP-rP1表达还与Rb基因状态有关,IGFBP-rP1低表达的乳腺癌磷酸化RB表达水平较高。RB基因调控IGFBP-rP1表达的确切机制也不清楚。近来有研究表明,RB、E2F可通过调控原癌基因EZH2的表达、影响其与DNMT1的结合来参与DNA甲基化过程。基于上述研究成果,本课题研究目标为明确RB、EZH2和DNMTs在IGFBP-rP1基因在结直肠癌中甲基化调控机制中的作用。我们利用western-blot和real-time PCR检测了实验室结直肠癌细胞株RKO、HT29、SW620和SW480中RB. EZH2和DNMTs表达情况,结果在这四株细胞中发现上述基因均有表达,所以采用RNA干扰(RNA interference, RNAi)技术沉默相关基因后,观察IGFBP-rP1转录水平表达改变情况,并利用BSP法检测IGFBP-rP1第一外显子甲基化状态的改变,旨在明确IGFBP-rP1甲基化调控通路。鉴于DNMTs在人类细胞甲基化中的作用,我们将DNMTs作为研究切入点,利用RNAi技术在IGFBP-rP1甲基化程度高且表达阴性的大肠癌RKO细胞系中分别沉默DNMT1、DNMT3b和DNMT3a,并建立稳定转染的单克隆细胞系,但是我们在上述基因单独沉默后并未检测到IGFBP-rP1表达改变。之后我们采取质粒共转来两两沉默DNMTs基因,我们发现在DNMT1和DNMT3b共同干扰的单克隆细胞系中IGFBP-rP1恢复了较弱的表达,染色质免疫共沉淀(Chromatin Immunoprecipitation, ChIP)结果显示DNMT1蛋白与IGFBP-rP1 DNA第一外显子直接结合而DNMT3b未检测到与IGFBP-rP1的直接作用。亚硫酸氢钠-测序法(Bisulfite sequencing PCR, BSP)检测IGFBP-rP1基因第一外显子甲基化改变情况,明确了DNMT1和DNMT3b共同参与了在IGFBP-rP1的甲基化调控中。我们还采用了MTT试验和流式细胞术检测了DNMT1和DNMT3b共同干扰可促进凋亡和抑制细胞增殖。结合IGFBP-rP1表达改变情况,我们推测IGFBP-rP1表达的恢复很可能参与了DNMT1和DNMT3b表达共同降低后引起的结直肠癌RKO细胞生物学行为的改变。接下来我们研究具有可调控DNMTs功能的EZH2和RB在IGFBP-rP1甲基化调控中是否发挥重要的作用。我们在有效沉默EZH2后并未发现IGFBP-rP1表达改变,而RB干扰后IGFBP-rP1有较明显的恢复表达,我们在IGFBP-rP1阴性表达的大肠癌细胞SW620中瞬时干扰RB也发现IGFBP-rP1表达的改变,我们将RB干扰后的RKO细胞建立稳定转染的单克隆细胞系用于检测IGFBP-rP1基因第一外显子甲基化改变情况。我们同样采用了MTT试验和流式细胞术检测了该单克隆细胞株在增殖能力和凋亡的改变,结果显示与对照组相比,RB干扰组凋亡率显著升高,细胞增殖能力下降。ChIP结果未发现RB与IGFBP-rP1存在直接的相互作用。我们发现在RB干扰的单克隆细胞株中DNMT1和DNMT3b均有明显下调,我们推测RB可能通过调控DNMTs家族来改变IGFBP-rP1基因第一外显子甲基化情况,进而改变IGFBP-rP1表达。通过对结直肠癌IGFBP-rP1基因甲基化调控机制的初步研究,我们得出以下结论：1. DNMT1和DNMT3b共同参与IGFBP-rP1基因甲基化调控,并且DNMT1蛋白与IGFBP-rP1基因有直接的相互作用。2.结直肠癌RKO细胞中未发现EZH2对IGFBP-rP1基因表达有明显的调控作用。3.RB参与IGFBP-rP1基因甲基化调控作用较明显,可能通过调控DNMTs家族来实现。更多还原

【Abstract】 Colorectal cancer (CRC) is one of the prevalent malignant tumors that threaten our health. In spite of the development of radiotherapy, chemotherapy and bio-targeted therapy, the mortality declines indistinctively. In China, the CRC incidence grows rapidly, especially in ZheJiang, ShangHai and JiangSu provinces. The low early detection rate is one of the important factors affecting the cancer therapy, so the early detection will promote the cancer prognosis.In 1999, our laboratory built three cDNA libraries using SSH (suppression subtractive hybridization):adenoma VS normal mucosa(A-N), adenocarcinoma VS adenoma(T-A), adenocarcinoma VS normal mucosa(T-N), and screened a series of genes with differential expression. IGFBP-rP1 (insulin-like growth factor binding protein-related protein 1) was screened from adenocarcinoma VS normal mucosa (T-N) cDNA subtraction library and is overexpressed in the colorectal adenocarcinoma tissue.IGFBP-rP1 is also named as IGFBP7 (Insulin-like growth factor binding protein 7), mac25 (meningioma associated cDNA 25), PSF (prostacyclin-stimulating factor), AGM (angiomodulin) and TAF (tumor-derived adhesion factor). It belongs to the IGFBP family and is a member of insulin-like growth factor (IGF) axis. IGFs have multiple functions regulating cell differentiation, proliferation and apoptosis. IGFBPs can bind IGFs and modulate the bioavailability of IGFs in the circulation. IGFBP-rPl, as a secreted protein, is widely detected in different types of body fluids, such as serum, urine, amniotic fluid and cerebrospinal fluid, and in various types of organs, such as thymus, prostate, bladder, liver, lung, stomach and colon. The majority view is that IGFBP-rPl plays a potential tumor suppressor role against human carcinogenesis with complex control mechanism, because it is down-regulated in most human tumors, including prostate carcinoma, breast carcinoma, liver carcinoma and meningioma, and overexpression of IGFBP-rPl can induce the apoptosis, arrest cell cycle and inhibit the proliferation of immortalized or malignat human cells. However, it has been reported that there is an opposite IGFBP-rPl expression pattern in several cancers, including colorectal cancer. This paradoxical phenomenon implied a reversible regulatory mechanism in this gene. Lin et al. found DNA methylation is the main regulatory mechanism underlying aberrant IGFBP-rPl expression in colorectal cancer, and aberrant methylation of the exon 1 of IGFBP-rP1 is inversely correlated with the expression of IGFBP-rP1 in colorectal cancer.Gene expression is regulated by two main mechanisms, genetics and epigenetics. Alterations of gene sequence are the typical situation of genetic modification, including gene amplifications, gene deletions, point mutations, loss of heterozygosity, chromosomal rearrangements, and overall aneuploidy, However, Epigenetic changes occur at a higher frequency than genetic changes and can be reversible upon treatment with pharmacological agents. Epigenetic modification is defined as a heritable, reversible change in gene expression that does not result from DNA sequence alterations, such as DNA methylation and demethylation, histone acetylation and deacetylation, non-coding RNA, etc. Aberrant DNA methylation is the most common type of epigenetic mechanism. It has become clear that hypermethylation of the 5’-flanking CpG islands silences gene expression and many suppressor genes are regulated by this pathway, abnormal DNA methylation is considered an early event in human carcinogenesis.DNA methylation is an epigenetic event in which DNA methyltransferases (DNMTs) cause the addition of a methyl group connected to the fifth carbon position of a cytosine residue in CpG dinucleotides. The mammalian DNMTs family mainly encompasses DNMT1, DNMT3a and DNMT3b. In mammals, DNMT3a and-3b are mainly responsible for establishing methylation at previously unmethylated sites, whereas DNMT1 is the major maintenance methyltransferase, reproducing existing methylation patterns during cell division. Selective targeting of DNMTs can result in gene-special demethylation and reexpression of many suppressor genes.The PcG protein EZH2 (Enhancer of Zeste homolog 2) is a histone methyltransferase associated with transcriptional repression. The Polycomb group protein EZH2 controls CpG methylation through direct physical contact with DNA methyltransferases. EZH2, as part of the PRC2/3 complexes, can physically recruit DNMTs to certain target-genes and that this process is essential for silencing the genes.Recent reports have demonstrated that the transcription of the Polycomb group (PcG) gene EZH2 is controlled by growth factors through the pRB-E2F pathway, which affects its interaction with DNMT1, thus this exerts influence of methylation. Downregulation of the potential suppressor gene IGFBP-rP1 is associated with inactivation of the retinoblastoma protein in human breast cancer. Then what is the relationship between RB and the aberrant DNA methylation of IGFBP-rPl and what is the role of DNMTs in the regulatory mechanism of the aberrant DNA methylation of IGFBP-rP1? It is our task to make them clear.In this study, we found that DNMTs, EZH2 and RB all express in four colorectal cell lines(RKO, HT29, SW620 and SW480). In order to disclose the regulatory mechanism of aberrant methylation of IGFBP-rPl, RNA interference (RNAi) technology was chosen to delete the expression of DNMTs, EZH2 and RB in colorectal RKO cell line without the endogenous IGFBP-rP1 expression.In view of the important role of DNMTs on the methylation in human cancer cells, our research was started from the study whether DNMTs take part in the aberrant DNA methylation of IGFBP-rP1. The expression of DNMT1, DNMT3a and DNMT3b were deleted respectively in colorectal RKO cell line and stable transfection cell lines were built with low level of DNMT1, DNMT3a, DNMT3b, but the restoration of IGFBP-rPl expression were not observed, so co-transfection was used to delete two of them at the same time, and the weak re-expression of IGFBP-rP1 was detectd in the monoclone cell line with decrease of DNMT1 and DNMT3b simultaneously, and the methylation status of IGFBP-rPl exon 1 was detected by MSP and BSP. The results of ChIP(Chromatin Immunoprecipitation) illustrate that not DNMT3b but the protein of DNMT1 can bind the IGFBP-rP1 exon 1 directly. Based the above results, we made it clear that cooperation of DNMT1 and DNMT3b plays an important role in the aberrant DNA methylation of IGFBP-rP1 in colorectal RKO cell line.Because EZH2 and RB were reported to control DNMTs recent years, our concern is wether they also are involved in the regulatory of aberrant DNA methylation of IGFBP-rP1. EZH2 was deleted effectively in RKO, but we didn’t observed the restoration of IGFBP-rP1 expression, while interference of RB gene in RKO induced the re-expression of IGFBP-rP1, The monoclone cell lines of selective deletion of RB were built to detect the methylation status of IGFBP-rP1 exon 1 by BSP. There is not interaction between RB and IGFBP-rP1 exon 1 in ChIP result, but we found the reduction of DNMT1 and DNMT3b in the RKO-RBi cell line.From the above results, we drew the following conclusions:1. Selective deletion of both DNMT1 and DNMT3b simultaneously in RKO cell can restore the IGFBP-rP1 expression, and DNMT1 binds the exon 1 of IGFBP-rP1 directly.2. Respective knockdowns of EZH2, DNMT1, DNMT3b and DNMT3a can not influence the expression of IGFBP-rP1.3. Decrease of RB in RKO cell can induce the hypomethylation of IGFBP-rP1 and restore the expression of IGFBP-rPl.4. Deletion of RB can result in the reduction of DNMT1 and DNMT3b, which maybe a pathway that RB regulates the methylation of the exon 1 of IGFBP-rP1.更多还原