【作者】 胡华梅；

【导师】 白云；

【作者基本信息】 第三军医大学 ， 遗传学， 2010， 硕士

【摘要】 食管癌是人类常见的恶性肿瘤之一,全世界每年约30万人死于食管癌,其中70%发生在我国,其中食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)约占病例总数的90%以上。因早期症状不明显并且缺乏有效的治疗措施,大部分食管癌患者就诊时已是中晚期,此时手术切除治疗的5年生存率仅为25%左右,是高致死性的疾病。因此寻找新的诊断治疗靶点对食管癌的早期诊断、治疗、预后以及预防具有重要的临床意义。DNA甲基化是表观遗传修饰的主要方式之一,主要发生于基因启动子附近的CpG岛。在许多人类肿瘤中都可以发现不同程度的DNA异常甲基化现象,表现为基因组整体甲基化水平降低,抑癌基因调控区域甲基化水平升高。目前认为,启动子区CpG岛超甲基化导致抑癌基因去表达是肿瘤中主要的表观遗传(epigenetics)改变之一,在许多恶性肿瘤的癌前病变中都发现了多个抑癌基因的CpG岛高甲基化。研究发现,食管癌癌变过程中抑癌基因启动子区域CpG岛的甲基化呈渐进性改变,启动子区CpG岛高甲基化在肿瘤的早期阶段、癌前阶段已存在,发生在细胞形态学改变之前,所以对基因甲基化状态进行检测,有望发现食管癌发生早期的生物标志。MicroRNA是一类21~25个碱基的小分子非编码RNA,存在于各种真核生物中。MicroRNA主要通过促进靶mRNA的降解或抑制其翻译过程而发挥调控作用,广泛参与细胞增殖、凋亡、代谢及分化等过程。MicroRNA的异常表达与肿瘤的发生、发展及演进有着极为密切的关系。Guo等通过芯片检测到在食管鳞癌中46种miRNA在肿瘤组织中的表达与癌旁正常组织具有显著性差异,表明miRNA在食管癌的发生和发展中起着重要作用。与其它基因一样,肿瘤细胞中miRNA的表达受到包括DNA甲基化修饰在内的多种机制的调控,而且miRNA基因甲基化状态的变化和肿瘤的类型及临床表型有关。由于不同组织来源的肿瘤存在各自特征性的miRNA表达谱,其甲基化状态既有共性也有各自的特性。迄今为止,还没有关于食管癌中相关miRNA的CpG岛甲基化状态的研究报道。因此,针对食管癌这一我国高发的恶性肿瘤,我们根据以往的研究资料,对在食管癌表达有差异的miRNA基因进行生物信息学分析,发现miR-34a、miR-34b/c、miR-424、miR-129-2的启动子区或周围都有CpG岛。食管癌中上述miRNA基因区域的甲基化状态是否具有特征性改变?成为本课题重点关注的问题。本研究将通过检测食管癌病例中miRNA的CpG甲基化状态,以期找到和食管癌相关的、潜在的早期诊断标志物。此外,miRNA-34是近年来发现的具有抑癌基因样功能的重要调控因子,是p53的靶基因,它的上调引起细胞周期阻滞和细胞凋亡,参与传统的P53的抑癌网络。miRNA在不同肿瘤中的表达改变具有细胞组织的差异,同时也与肿瘤发生和进展的进程相关。因此,本研究将进一步明确在食管癌中,CpG甲基化改变是否参与miR-34表达的调控,在此基础上,尝试探讨干预甲基化状态对miR-34表达及其靶基因的影响,为以后进一步将miRNA分子作为早期干预治疗的靶点提供线索。第一部分肿瘤相关miRNA基因的CpG岛甲基化状态与食管癌的相关性研究方法:通过生物信息学分析和文献数据库挖掘,选取启动子区域具有CpG岛的肿瘤相关miRNA分子miR-34a、miR-34b/c、miR-424、miR-129-2为研究对象。采用BSP和MSP的方法检测食管癌肿瘤组织、远癌组织、食管癌细胞株(KYSE150、KYSE 450、EC109、EC9706)的甲基化状态。研究结果:miR-424在肿瘤组织/细胞株和远癌组织中都是甲基化的,没有差异。食管癌细胞株(EC109、EC9706、KYSE450、KYSE150)中miR-34a、miR-34b/c、miR424、miR129-2都是甲基化的。肿瘤组织中的甲基化率miR-34a为66.7%(36/54),miR-34b/c为40.7%(22/54), miR129-2为100%(27/27) ;远癌组织中miR-34a为26.7%(8/30),miR-34b/c为0(0/30),miR129-2为0(0/13),经统计学分析表明三种miRNA在肿瘤组织和细胞株中的甲基化率显著高于远癌组织。miR-34a和miR-34b/c的甲基化率与肿瘤的分化情况、临床分期、淋巴结转移情况等临床表型参数无关(p>0.05)。其中miR-129-2在检测的27例食管癌中全部甲基化而远癌组织没有甲基化,提示miR-129-2的甲基化状态可能成为食管癌细胞的特征性标志,可能成为潜在的早期诊断靶点。第二部分DNA甲基化对食管癌miR-34表达及其靶基因的影响研究方法:1.选择miR-34为进一步研究靶点,TaqMan Real-time PCR方法检测24例肿瘤组织的miR-34的表达情况,比较甲基化组和非甲基化组miR-34的表达差异。2.利用去甲基化试剂5-Aza-CdR处理食管癌细胞株KYSE150、KYSE450,采用甲基化特异性PCR(MSP)检测甲基化逆转情况,Real-time PCR检测用药前后miR-34的表达。3. Western Blot免疫印迹法检测食管癌细胞株5-Aza-CdR处理前后miR-34a靶基因Bcl-2的表达变化情况。研究结果:1. 24例食管癌肿瘤组织中,甲基化组miR-34a、miR-34b的表达水平显著性低于非甲基化组。(p<0.05)2.甲基化酶抑制剂5-Aza-CdR处理食管癌细胞株后,MSP分析发现miR-34-a,b启动子区域发生去甲基化,Real-time PCR检测处理前后miR-34的表达,结果显示miR-34的表达随着甲基化状态的丢失而上调。3. Western Blot免疫印迹法分析显示,未加药处理的KYSE150、KYSE450细胞株在分子量为26KD的位置均有明显条带;而5-Aza-CdR处理的KYSE150没有条带,KYSE450的条带较其未加药处理前弱。提示消除miR-34a的甲基化状态可以提高miR-34a的表达,进而抑制原癌基因Bcl-2的产生。更多还原

【Abstract】 Esophageal cancer is one of the most common malignancy worldwide, more than 300,000 people died of this cancer each year. Among these cases, 70% occurred in China and more than 90% of these are esophageal squamous cell carcinoma. As an extremely fatal disease, esophageal cancer is not easy to diagnose at the early stage. Most cases were found at later stage and surgical operation had to be carried out. However, the 5-years survival rate is only 25%.Therefore, it is very important to find new target for early diagnosis and treatment.DNA methylation is one of the main manners of epigenetics modification, it happens mainly in the CpG island around the gene promoter region. Aberrant DNA methylation has been reported in many different types of cancer in human. In tumor the methylation is low in most regions but high in the regulation regions of anti-oncogenes. The down regulation of anti-oncogenes expression caused by the CpG hypermethylation in their promoter region is said to be one of the main epigenetic changes in tumor. The CpG hypermethylation have been found in many anti-oncogenes promoter regions in many tumor types. There are data showing that the CpG methylation changing in the promoter region is a gradual progress during the oncogenesis. The Hypermethylation of CpG island in promoter region begins at the very early stage of oncogenesis and can be detected before the tumor formation. So as for the early diagnosis biomarker, to find the oncogenesis as early as possible, it is much better to assay the methylation modification before the cell morphology changing can be detected.microRNAs are a group of 21-25nt small non-coding RNA, seeing in many eukaryotic organisms. microRNAs play important regulation roles in cell proliferation, cell apoptosis, differentiation and cell metabolism by degrading their target mRNAs or inhibit their translation. The deregulation of microRNAs has great influence on oncogenesis, tumor development and differentiation. In 2008,using micro-array analysis, Guo et al and Feber et al found many up-regulation and down-regulation miRNAs related with ESCC. These data suggested miRNAs were also important in oncogenesis and development of ESCC. As coding genes, the expression of miRNAs are regulated by many kinds of machinisms. Among them, DNA methylation is very important. For example, Furuta et al found the expression of miR-124 and miR-203 were regulated by DNA methylation, such methylation modification is associated with hepatic carcinoma. Since there are different miRNA expression pattern in different types of tumors, DNA methylation pattern of miRNAs have respective characteristics besides of commonness. While there is no study reported on the methylation status of those ESCC associated miRNAs so far.It is of great interest to know if the methylation status of tumor assocated miRNAs in ESCC changed or not. To answer this question, we detected the CpG methylation status in ESCC tissues and paraneoplastic tissues in this study. After that, we compared and statistically analyzed the results between ESCC group and normal tissues group to unfold new biomarkers for ESCC early detection.Besides the first aim, miRNA34a/b/c are newly discovered anti-oncogenes. As a p53 target and part of classical p53 anti tumor network, its’up-regulation is important in cell apoptosis and cell cycle blockade. There are evidences showing that miR-34a expresses specific in different tissues. To provide clues on using miRNA methylation status as potential therapy target, we studied the influence of methylation interference drugs on the expression of miR-34a and its target gene in second part of this study. Part 1: Detection of DNA methylation around ESCC related miRNA gene promoter region.Method: After bioinformatics analysis and data mining, cancer related miRNAs with CpG island in their promoter region (miR-34a、miR-34b/c、miR-424、miR-129-2) were choose for further study. The methylation status around their miRNAs were analyzed with BSP and MSP in ESCC and paraneoplastic tissues, as well as 4 ESCC cell lines (KYSE150、KYSE 450、EC109、EC9706)Results: The CpG sites in miR-424 promoter region were methylated in all tested ESCC and paraneoplastic tissues, and also in the 4 cell lines. While for another three miRNAs, the CpG sites were predominant methylated in ESCC tissues, with 66.7%(36/54) in miR-34a, 40.7%(22/54)in miR-34b/c and 100%(27/27) in miR129-2. (P<0.05 for all comparisons). The methylation status around miR34a/b/c has nothing to do with the differentiation, clinical stages and lymph metastasis. These data suggested miR-34a/b/c and miR-129-2 may be able to server as diagnosis bio-maker for ESCC.Part 2: Interfering DNA methylation increased miR-34a expression and down regulated the expression of ongene Bcl-2.Method: 1. The expression of miR-34a in ESCC tissues were detected with Taq-Man qPCR. The difference of miR-34a expression were compared between methylated group and unmethylated group.2. ESCC cell lines KYSE150 and KYSE450 were treated with 5-Aza-CdR. The methylation statuses were examined after treatment with MSP. The expression of miR-34a was evaluated before and after the treatment. And the protein expression of Bcl-2, a target gene of miR-34a was assessed with western blot before and after treatment.Results: 1.The expression level of miR-34a and miR-34b in methylation group were significant higher than the un-methylation group. (p<0.05)2. After 5-Aza-CdR treatment, CpG sites in miR-34a,b promoter region were demethylated. Real time PCR found the expression of miR-34a was increased after the treatment. The western blot shown the expression of Bcl-2 was down regulated after treatment.Conclusions: In this study, the methylation status of CpG sites in miR-34a, miR-34b/c and miR129-2 were significant higher in ESCC tissue than in paraneoplastic tissues, suggested the CpG methylation in miR-34a and miR-129-2 region might be able to server as bio-markers for ESCC diagnosis. Preliminary data presented here suggested that targeting those methylation statuses with appropriate drugs has the potential to cure the ESCC disease.更多还原