CLDN11基因甲基化与结直肠癌转移的相关性研究
发布时间：2018-07-23 19:14
【摘要】：目的结直肠癌(colorectal cancer)是消化系统中最常见的恶性肿瘤之一,是正常肠黏膜上皮细胞在多种致病因素的共同作用下恶性转变的结果。目前进展期结直肠癌患者5年生存率仅有30%,其低生存率的问题亟待解决。结直肠癌复发转移正是当前结直肠癌低生存率主要原因。因此,深入理解结直肠癌发生转移的机制是解决结直肠癌患者低生存率的关键。随着肿瘤研究的深入,以DNA甲基化为重要组分的表观遗传学修饰在结直肠癌发生发展过程中发挥的作用逐步得到重视。研究报道,DNA甲基化发生于结直肠癌早期,甚至早于遗传学改变,在早期诊断筛查结直肠癌方面具有重要的临床应用价值。由此,研究结直肠癌早期发生的异常甲基化基因修饰中,是否存在能够预测结直肠癌转移或者患者生存率的风险因素,通过干预基因甲基化水平,是否其对癌细胞迁移运动能力产生影响,有助于深入结直肠癌复发转移的发病机制,实时监测结直肠癌发展动态,同时为结直肠癌治疗新策略提供理论基础。因此,本研究主要分析结直肠癌发生过程中异常的DNA甲基化修饰,同时分析其与结直肠癌转移的关系。方法1.应用Illumina humanmethylaiton 450K芯片(HM450K)检测分析结直肠癌与癌旁组织间差异甲基化基因;利用DAVID在线网站(https://david.ncifcrf.gov/)注释分析差异甲基化基因的生物信息学功能,如信号通路、生物学功能等。基于HM450K芯片结果,我们发现细胞黏附信号通路(cellular adhesion moleculars,CAMs)中紧密连接蛋白-11(CLDN11)在结直肠癌组织明显高甲基化,由此我们将研究CLDN11甲基化在结直肠癌发生发展过程中发挥的作用;2.随后我们应用荧光定量甲基化特异性聚合酶链式反应(quantitative methylation specific polymerase chain reaction,qMSP)在125对结直肠癌组织及癌旁组织中检测CLDN11甲基化水平;并结合患者临床病理信息,分析CLDN11甲基化与临床特征相关性;3.应用双荧光素酶报告基因实验,分析CLDN11启动子片段是否具有启动基因表达功能;4.应用荧光定量逆转录-聚合酶链式反应(quantitative reverse transcriptionpolymerase chain reaction,qRT-PCR)检测100对结直肠癌组织及对应的癌旁组织中CLDN11 m RNA表达水平,分析CLDN11甲基化和表达相关性;5.应用qMSP和qRT-PCR检测正常肠黏膜上皮细胞株(NCM460)和4株结直肠癌细胞株(HCT116,COLO205,SW620和HT29)中CLDN11甲基化和表达水平情况;6.配置不同浓度5-aza-dC去甲基化药物改变结直肠癌细胞CLDN11甲基化水平,应用qMSP和qRT-PCR观察CLDN11甲基化和表达变化,确定最佳药物浓度;7.应用Transwell迁移实验,观察CLDN11甲基化改变对结直肠癌细胞株迁移能力的影响;8.通过公共肿瘤数据库TCGA(https://cancergenome.nih.gov/),整合CLDN11甲基化数据、表达数据和患者临床病理信息,大样本验证CLDN11甲基化与表达相关性及与结直肠癌患者的无复发生存期相关性;结果1.根据HM450K芯片结果,共筛选出差异甲基化位点4047个,覆盖1725个参考基因。基于HM450K芯片注释分析结果,我们发现细胞黏附信号通路(cellular adhesion moleculars,CAMs)中CLDN11在结直肠癌组织明显高甲基化;2.与癌旁组织相比,结直肠癌组织中CLDN11甲基化率显著增高(P=3.71E-23)。结合患者临床数据分析后发现,发生淋巴结转移结直肠癌患者组织中,CLDN11甲基化率高于未发生淋巴结转移的结直肠癌患者(P=0.01);3.双荧光素酶报告基因实验发现,插入的被检的CLDN11启动子区域片段具有启动表达功能(P=0.002);4.q RT-PCR结果显示,相较与癌旁组织,CLDN11 m RNA表达水平在结直肠癌组织中显著降低(P=0.002);并且CLDN11甲基化与表达呈负性相关(r=-0.19,P=0.04),提示CLDN11启动子甲基化是其表达降低的原因之一;5.在我们培养的4株结直肠癌细胞中,HCT116、COLO205和HT29结直肠癌细胞株中CLDN11的甲基化率高于NCM460,而SW620中甲基化率明显低于NCM460;同时我们检测细胞中CLDN11 mRNA表达水平,结果发现HCT116、COLO205和HT29结直肠癌细胞株中CLDN11 mRNA表达水平低于NCM460,而SW620细胞中CLDN11 mRNA表达水平高于NCM460;该结果同样提示CLDN11启动子甲基化是其表达降低的原因之一;6.基于结直肠癌细胞中CLDN11甲基化和表达情况,我们最后选择HCT116细胞株进行5-aza-dC去甲基化药物处理。qMSP和qRT-PCR实验结果发现,使用9μmol/L 5-aza-dC处理HCT116时,CLDN11甲基化率改变最显著;7.Transwell实验证明结直肠癌细胞株降低CLDN11甲基化水平后,细胞迁移能力明显减弱;8.基于TCGA甲基化和表达数据结果证实,结直肠癌组织中CLDN11甲基化程度与其表达成负性相关(r=-0.21,P=0.000035);结合患者临床数据显示,CLDN11高甲基化状态与结直肠癌低无复发生存期(relaspe free survival,RFS)相关(Log rank P=0.04)。结论经过实验证实CLDN11甲基化参与结直肠癌转移。并且结合公共数据库大样本数据分析显示,CLDN11甲基化是预示结直肠癌复发潜在的预后评估分子标志物。
[Abstract]:Objective colorectal cancer (colorectal cancer) is one of the most common malignant tumors in the digestive system. It is the result of the malignant transformation of normal intestinal epithelial cells under the joint action of various pathogenic factors. The 5 year survival rate of the patients with colorectal cancer is only 30%, and the problem of low survival rate is urgent to be solved. It is the main reason for the low survival rate of colorectal cancer. Therefore, a deep understanding of the mechanism of metastasis of colorectal cancer is the key to solving the low survival rate of colorectal cancer patients. With the development of cancer research, the role of epigenetic modification with DNA methylation as an important component in the development of colorectal cancer is gradually paid attention to. DNA methylation, which occurs early in colorectal cancer and even earlier than genetic changes, is of important clinical value in early diagnosis and screening of colorectal cancer. Therefore, it is possible to study the risk of predicting colorectal cancer metastasis or the risk of survival in colorectal cancer early onset of abnormal methylation gene modification. Factors, by interfering with the level of gene methylation and whether it affects the migration and movement of cancer cells, can help to deepen the pathogenesis of recurrence and metastasis of colorectal cancer, monitor the development of colorectal cancer in real time, and provide a theoretical basis for the new strategy of colorectal cancer treatment. Normal DNA methylation modification and analysis of its relationship with metastasis of colorectal cancer. Method 1. Illumina humanmethylaiton 450K chip (HM450K) was used to detect the differential methylation genes between colorectal cancer and para cancerous tissues, and the bioinformatics work of differentially methylated genes was analyzed by DAVID online website (https://david.ncifcrf.gov/) annotation. Yes, such as signal pathways, biological functions, and so on. Based on the results of HM450K chip, we found that the close connexin -11 (CLDN11) in the cell adhesion signaling pathway (cellular adhesion moleculars, CAMs) is highly methylation in the colorectal cancer tissue, thus we will study the role of CLDN11 methylation in the development of colorectal cancer; 2. Then we used quantitative methylation specific polymerase chain reaction (qMSP) to detect the level of CLDN11 methylation in 125 colorectal cancer tissues and para cancerous tissues, and combined with the clinicopathological information of the patients to analyze the correlation between CLDN11 methylation and clinical features; 3. The luciferase reporter gene experiment was used to analyze whether the CLDN11 promoter fragment had the function of promoter gene expression. 4. the expression level of CLDN11 m RNA in 100 colorectal tissues and corresponding para cancerous tissues was detected by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Analysis of CLDN11 methylation and expression correlation; 5. qMSP and qRT-PCR were used to detect the CLDN11 methylation and expression levels in normal intestinal mucosal epithelial cell line (NCM460) and 4 colorectal cancer cell lines (HCT116, COLO205, SW620 and HT29); 6. configuration of different concentrations of 5-aza-dC normethylation drugs to change the CLDN11 methylation level of colorectal cancer cells, QMSP and qRT-PCR were used to observe the CLDN11 methylation and expression changes and determine the best drug concentration. 7. the effect of CLDN11 methylation on the migration ability of colorectal cancer cell lines was observed by Transwell migration test. 8. through the public tumor database TCGA (https://cancergenome.nih.gov/), the data of CLDN11 methylation, the expression of data and the patient's presence were observed. Bed pathological information, large samples verified the correlation between CLDN11 methylation and expression and the recurrence free survival of patients with colorectal cancer. Results 1. according to the results of HM450K chip, 4047 different methylation sites were screened and 1725 reference genes were covered. Based on the HM450K chip annotation analysis, we found cell adhesion signaling pathway (cellul CLDN11 in AR adhesion moleculars, CAMs) was significantly methylation in colorectal cancer tissue; 2. compared with para cancerous tissue, the CLDN11 methylation rate was significantly higher in colorectal cancer tissues (P=3.71E-23). The incidence of CLDN11 methylation in colorectal cancer patients was higher than that of non lymph node metastases in patients with lymph node metastases. Migration of colorectal cancer patients (P=0.01); 3. double luciferase reporter gene experiment found that the inserted CLDN11 promoter region fragment had the function of starting expression (P=0.002); 4.q RT-PCR results showed that the expression level of CLDN11 m RNA was significantly lower in the colorectal carcinoma than that of the paracancerous tissue (P=0.002); and CLDN11 methylation and expression. Negative correlation (r=-0.19, P=0.04), suggesting that CLDN11 promoter methylation is one of the reasons for its reduction. 5. of the 4 colorectal cancer cells in our culture, the methylation rate of CLDN11 in the HCT116, COLO205 and HT29 colorectal cancer cell lines is higher than NCM460, and the methylation rate in SW620 is significantly lower than that of NCM460; meanwhile, CLDN11 mR in the cells is detected. The expression level of NA showed that the expression level of CLDN11 mRNA in the HCT116, COLO205 and HT29 colorectal cancer cell lines was lower than that of NCM460, and the CLDN11 mRNA expression level in SW620 cells was higher than that of NCM460. The results also suggested that the methylation of the CLDN11 promoter was one of the reasons for its decrease in expression, and the 6. base was methylated and expressed in the colorectal cancer cells. We finally selected the HCT116 cell line for the 5-aza-dC methylation drug treatment.QMSP and qRT-PCR results, and found that the CLDN11 methylation rate was the most significant change when using 9 mu mol/L 5-aza-dC to treat HCT116, and the 7.Transwell test showed that the cell migration ability of colorectal cancer cell lines decreased significantly after the reduction of CLDN11 methylation water, and 8. based on TCGA. Methylation and expression data showed that the degree of CLDN11 methylation in colorectal cancer tissues was negatively correlated with their expression (r=-0.21, P=0.000035). Combined with clinical data, CLDN11 methylation was associated with low relapse free survival (relaspe free survival, RFS) in colorectal cancer (Log rank P=0.04). Conclusion CLDN11 was experimentally confirmed. Methylation participates in the metastasis of colorectal cancer. And combined with large sample data from the public database, CLDN11 methylation is a potential prognostic molecular marker for the recurrence of colorectal cancer.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2140384
[Abstract]:Objective colorectal cancer (colorectal cancer) is one of the most common malignant tumors in the digestive system. It is the result of the malignant transformation of normal intestinal epithelial cells under the joint action of various pathogenic factors. The 5 year survival rate of the patients with colorectal cancer is only 30%, and the problem of low survival rate is urgent to be solved. It is the main reason for the low survival rate of colorectal cancer. Therefore, a deep understanding of the mechanism of metastasis of colorectal cancer is the key to solving the low survival rate of colorectal cancer patients. With the development of cancer research, the role of epigenetic modification with DNA methylation as an important component in the development of colorectal cancer is gradually paid attention to. DNA methylation, which occurs early in colorectal cancer and even earlier than genetic changes, is of important clinical value in early diagnosis and screening of colorectal cancer. Therefore, it is possible to study the risk of predicting colorectal cancer metastasis or the risk of survival in colorectal cancer early onset of abnormal methylation gene modification. Factors, by interfering with the level of gene methylation and whether it affects the migration and movement of cancer cells, can help to deepen the pathogenesis of recurrence and metastasis of colorectal cancer, monitor the development of colorectal cancer in real time, and provide a theoretical basis for the new strategy of colorectal cancer treatment. Normal DNA methylation modification and analysis of its relationship with metastasis of colorectal cancer. Method 1. Illumina humanmethylaiton 450K chip (HM450K) was used to detect the differential methylation genes between colorectal cancer and para cancerous tissues, and the bioinformatics work of differentially methylated genes was analyzed by DAVID online website (https://david.ncifcrf.gov/) annotation. Yes, such as signal pathways, biological functions, and so on. Based on the results of HM450K chip, we found that the close connexin -11 (CLDN11) in the cell adhesion signaling pathway (cellular adhesion moleculars, CAMs) is highly methylation in the colorectal cancer tissue, thus we will study the role of CLDN11 methylation in the development of colorectal cancer; 2. Then we used quantitative methylation specific polymerase chain reaction (qMSP) to detect the level of CLDN11 methylation in 125 colorectal cancer tissues and para cancerous tissues, and combined with the clinicopathological information of the patients to analyze the correlation between CLDN11 methylation and clinical features; 3. The luciferase reporter gene experiment was used to analyze whether the CLDN11 promoter fragment had the function of promoter gene expression. 4. the expression level of CLDN11 m RNA in 100 colorectal tissues and corresponding para cancerous tissues was detected by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Analysis of CLDN11 methylation and expression correlation; 5. qMSP and qRT-PCR were used to detect the CLDN11 methylation and expression levels in normal intestinal mucosal epithelial cell line (NCM460) and 4 colorectal cancer cell lines (HCT116, COLO205, SW620 and HT29); 6. configuration of different concentrations of 5-aza-dC normethylation drugs to change the CLDN11 methylation level of colorectal cancer cells, QMSP and qRT-PCR were used to observe the CLDN11 methylation and expression changes and determine the best drug concentration. 7. the effect of CLDN11 methylation on the migration ability of colorectal cancer cell lines was observed by Transwell migration test. 8. through the public tumor database TCGA (https://cancergenome.nih.gov/), the data of CLDN11 methylation, the expression of data and the patient's presence were observed. Bed pathological information, large samples verified the correlation between CLDN11 methylation and expression and the recurrence free survival of patients with colorectal cancer. Results 1. according to the results of HM450K chip, 4047 different methylation sites were screened and 1725 reference genes were covered. Based on the HM450K chip annotation analysis, we found cell adhesion signaling pathway (cellul CLDN11 in AR adhesion moleculars, CAMs) was significantly methylation in colorectal cancer tissue; 2. compared with para cancerous tissue, the CLDN11 methylation rate was significantly higher in colorectal cancer tissues (P=3.71E-23). The incidence of CLDN11 methylation in colorectal cancer patients was higher than that of non lymph node metastases in patients with lymph node metastases. Migration of colorectal cancer patients (P=0.01); 3. double luciferase reporter gene experiment found that the inserted CLDN11 promoter region fragment had the function of starting expression (P=0.002); 4.q RT-PCR results showed that the expression level of CLDN11 m RNA was significantly lower in the colorectal carcinoma than that of the paracancerous tissue (P=0.002); and CLDN11 methylation and expression. Negative correlation (r=-0.19, P=0.04), suggesting that CLDN11 promoter methylation is one of the reasons for its reduction. 5. of the 4 colorectal cancer cells in our culture, the methylation rate of CLDN11 in the HCT116, COLO205 and HT29 colorectal cancer cell lines is higher than NCM460, and the methylation rate in SW620 is significantly lower than that of NCM460; meanwhile, CLDN11 mR in the cells is detected. The expression level of NA showed that the expression level of CLDN11 mRNA in the HCT116, COLO205 and HT29 colorectal cancer cell lines was lower than that of NCM460, and the CLDN11 mRNA expression level in SW620 cells was higher than that of NCM460. The results also suggested that the methylation of the CLDN11 promoter was one of the reasons for its decrease in expression, and the 6. base was methylated and expressed in the colorectal cancer cells. We finally selected the HCT116 cell line for the 5-aza-dC methylation drug treatment.QMSP and qRT-PCR results, and found that the CLDN11 methylation rate was the most significant change when using 9 mu mol/L 5-aza-dC to treat HCT116, and the 7.Transwell test showed that the cell migration ability of colorectal cancer cell lines decreased significantly after the reduction of CLDN11 methylation water, and 8. based on TCGA. Methylation and expression data showed that the degree of CLDN11 methylation in colorectal cancer tissues was negatively correlated with their expression (r=-0.21, P=0.000035). Combined with clinical data, CLDN11 methylation was associated with low relapse free survival (relaspe free survival, RFS) in colorectal cancer (Log rank P=0.04). Conclusion CLDN11 was experimentally confirmed. Methylation participates in the metastasis of colorectal cancer. And combined with large sample data from the public database, CLDN11 methylation is a potential prognostic molecular marker for the recurrence of colorectal cancer.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前2条
1 Shao-Qin Chen;Zhi-Hua Chen;Su-Yong Lin;Qi-Bao Dai;Leng-Xi Fu;Rui-Qing Chen;;KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients[J];World Journal of Gastroenterology;2014年29期
2 rpád V Patai;Béla Molnár;Zsolt Tulassay;Ferenc Sipos;;Serrated pathway:Alternative route to colorectal cancer[J];World Journal of Gastroenterology;2013年05期
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