高通量分析人食管鳞癌甲基化差异基因的相关研究

发布时间：2019-03-08 14:49

【摘要】：食管癌是世界上最常见的恶性肿瘤之一,其发病率占全球肿瘤发病率的第八位,死亡率占癌症相关死亡率的第六位。我国食管癌发病率占世界首位,是食管鳞状细胞癌(Esophageal squamous cell carcinoma, ESCC)高发地区,约占全球范围内的70%。近年来,其发病率虽有所下降,但ESCC的长期预后仍然较差,术后五年生存率只有15%～25%。DNA甲基化异常改变被认为是ESCC发生发展的常见分子机制之一,主要特征是全基因组低甲基化和位点特异CpG岛高甲基化。进一步了解DNA异常甲基化如何调控ESCC的发生与发展,寻找ESCC甲基化异常改变分子标志物,在指导人们对ESCC患者预后判断以及制定有效治疗方案等方面具有重要临床意义。在本文的第一部分研究中,我们用简化的、具有代表性的DNA甲基化测序(RRBS)及基因表达谱芯片高通量分析技术分析了7例ESCC患者癌及其癌旁组织DNA甲基化水平和mRNA表达水平。先对ESCC和其癌旁差异甲基化基因进行功能富集分析,差异基因主要功能富集在分子连接与调节、细胞组成与调节、催化活性与代谢调节等。另外,细胞运动、物质运输以及细胞间相互作用相关基因发生较大改变。接着,结合前期表达谱芯片结果,筛出位于启动子区DNA甲基化与基因表达水平呈负相关基因。为了筛选ESCC发生发展过程中特异的甲基化改变基因,挑选前10个具有显著差异的基因,结合功能和文献检索,筛选出甲基化升高同时表达降低基因Cyclin Y-like l(CCNYL1)进行后续研究,经过癌症基因组图谱数据库(TCGA)预测获得其在ESCC中未有突变、缺失和扩增等改变。进一步我们用硫化测序PCR(BSP)技术在17对ESCC组织中验证DNA甲基化水平和Real-time PCR技术在10对冰冻ESCC组织中验证mRNA表达水平,结果与RRBS和表达谱芯片结果一致,CCNYL1在ESCC组织中呈高甲基化低表达。经统计学分析,在ESCC中CCNYL1基因DMR处CpG位点13、CpG位点16、CpG位点17、CpG位点18和CpG位点19呈现高甲基化。与人食管上皮永生化细胞株相比,CCNYL1在ESCC细胞株中DNA甲基化水平显著升高,mRNA表达水平降低。然后,通过体外实验在ESCC细胞株中过表达CCNYL1基因,结果显示其对癌细胞增殖有抑制作用。最后,我们采用表观遗传治疗法,经5-杂氮脱氧胞苷(5-aza-dC)处理ESCC细胞株后,CCNYL1基因启动子区DNA甲基化水平降低,而mRNA表达升高,说明甲基化直接调节CCNYL1的表达。另外DMR中CpG位点13、16、17和19甲基化水平显著降低,为肿瘤治疗提供分子基础。所以,CCNYL1基因高甲基化可能参与ESCC的发生,其DMR可能为肿瘤治疗提供新靶点。在本文第二部分的研究中,筛选前期高通量分析获得ESCC淋巴结转移和无淋巴结转移组中甲基化程度与表达水平负相关基因,DMR位于启动子区的差异基因共5个,其中ODC1已在食管癌中有报道,PHB2、 PCMT1、 ARHGEF4在癌症中也有许多研究,只有HSD11B1L研究较少,本部分重点研究HSD11B1L在ESCC发生发展中作用。经验证HSD11B1L在ESCC组织及细胞株中DNA甲基化水平下降(P0.001),与mRNA表达成负相关,并且在淋巴结转移的ESCC组织中,其甲基化水平低于无淋巴结转移的组织(P0.001)。另外,在ESCC细胞株中,敲降HSD11B1L影响其恶性程度,能抑制细胞的增殖和侵袭能力。为探索HSD11B1L甲基化频率与临床病理参数之间的相关性,另外增加20例淋巴结转移和20例无淋巴结转移的石蜡包埋ESCC组织用BSP检测HSD11B1L甲基化水平,结果表明,HSD11B1L在淋巴结转移的组织中甲基化水平低(P0.001),提示HSD11B1L甲基化水平低的患者更容易发生转移。在癌组织中HSD11B1L的甲基化水平与肿瘤分化程度和淋巴结转移有关联性(P=0.039,P=0.004)。将ESCC组织进行Kaplan-Meier生存分析,得到的结果是HSD11B1L异常低甲基化的ESCC病人预后较差(P=0.003),多变量Cox回归生存模型分析结果显示HSD11B1L的甲基化是ESCC病人的一个独立的预后因素(P=0.01)。所以,HSD11B1L异常低甲基化在ESCC发生发展中发挥重要作用,是一个独立的预后因素。
[Abstract]:Esophageal cancer is one of the most common malignant tumors in the world. The incidence of esophageal cancer is the eighth of the global tumor incidence, and the mortality rate is the sixth of the cancer-related mortality. The incidence of esophageal cancer in China is the first in the world, and it is an area of high incidence of esophageal squamous cell carcinoma (ESCC), accounting for about 70% of the world. In recent years, the incidence of ESCC has decreased, but the long-term prognosis of ESCC is still poor. The five-year survival rate of ESCC is only 15% ~ 25%. The abnormal changes of DNA methylation are considered one of the most common molecular mechanisms for the development of ESCC. The main features are the low methylation of the whole genome and the high methylation of the site-specific CpG island. It is of great significance to find out how to regulate and control the occurrence and development of ESCC, to find the change of molecular markers in ESCC methylation, and to guide people to judge the prognosis of ESCC and to develop effective treatment plan. In the first part of this paper, we used a simplified, representative DNA methylation sequencing (RRBS) and high-throughput analysis of gene expression profiles to analyze the level of DNA methylation and the level of mRNA expression in 7 ESCC patients and their adjacent tissues. The functional enrichment analysis of the differentially methylated genes of ESCC and its adjacent cancer is carried out. The main function of the differential gene is the molecular connection and regulation, the composition and regulation of the cells, the catalytic activity and the regulation of metabolism, etc. In addition, there is a great change in cell movement, material transport, and inter-cell interaction-related genes. Then, the DNA methylation of the promoter region and the gene expression level were negatively correlated with the results of the earlier expression spectrum chip. In order to screen the specific methylation change gene in the development of ESCC, the first ten genes with significant difference, combined with function and literature search, were selected to screen out the simultaneous expression of the reduced gene Cyclin Y-like l (CCNYL1). A cancer genomic map database (TCGA) was used to predict the absence of mutation, deletion and amplification in ESCC. Further, we validated the level of DNA methylation and the real-time PCR in the ESCC tissue using a vulcanization sequencing PCR (BSP) technique to verify the level of mRNA expression in the 10-to-frozen ESCC tissue, and the results were consistent with the results of the RRBS and the expression profile, and the CCNYL1 was highly methylated and low in the ESCC tissue. By statistical analysis, the CpG sites 13, the CpG sites 16, the CpG sites 17, the CpG sites 18, and the CpG sites 19 of the CCNYL1 gene DMR were hypermethylated in the ESCC. Compared with the immortalized cell line of human esophageal epithelium, the level of DNA methylation of CCNYL1 in the ESCC cell line was significantly increased and the level of mRNA expression was decreased. Then, the CCNYL1 gene is overexpressed in the ESCC cell strain by in vitro experiment, and the result shows that the CCNYL1 gene has the inhibition effect on the proliferation of the cancer cells. Finally, we used epigenetic therapy to reduce the level of DNA methylation in the promoter region of the CCNYL1 gene, and the expression of the mRNA was increased, indicating the direct regulation of the expression of the CCNYL1. In addition, that methylation level of the CpG sites 13,16,17 and 19 in the DMR is obviously reduce, and the molecular basis for tumor treatment is provided. Therefore, the high methylation of the CCNYL1 gene may be involved in the occurrence of ESCC, and its DMR may provide a new target for tumor therapy. In the second part of this paper, there was a negative correlation between the degree of methylation and the level of expression in the lymph node metastasis of the ESCC and the level of expression of the non-lymph node metastasis group in the early stage of screening, and the DMR in the promoter region was 5, among which ODC1 had been reported in the esophageal cancer, PHB2, PCMT1, ARHGEF4 also has a number of studies in the treatment of cancer, and only the ADuC11B1L is less research, and this part focuses on the role of B11B1L in the development of ESCC. The level of DNA methylation in ESCC and cell lines decreased (P 0.001) and negative correlation with the expression of mRNA, and the methylation level was lower in the ESCC of the lymph node metastasis than that of the non-lymph node metastasis (P 0.001). In addition, in the ESCC cell line, the knock-down antigen 11B1L affects the degree of malignancy and can inhibit the proliferation and the invasion ability of the cell. In order to explore the correlation between the methylation frequency and the clinical pathological parameters of B11B1L,20 cases of lymph node metastasis and 20 paraffin-embedded ESCC tissues with no lymph node metastasis were used to detect the methylation level of B11B1L. The results showed that the methylation level of B11B1L in the lymph node metastasis was low (P 0.001). It is suggested that the patients with low methylation level of the CYP11B1L can be more easily transferred. The methylation level of p11B1L in cancer tissues was associated with the degree of tumor differentiation and lymph node metastasis (P = 0.039, P = 0.004). The results of the Kaplan-Meier survival analysis of ESCC were poor (P = 0.003), and the analysis of multivariate Cox regression survival model showed that the methylation of B11B1L was an independent prognostic factor for ESCC patients (P = 0.01). Therefore, the abnormal low methylation of P11B1L plays an important role in the development of ESCC and is an independent prognostic factor.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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