CLDN11基因甲基化與結(jié)直腸癌轉(zhuǎn)移的相關(guān)性研究
發(fā)布時間：2018-07-23 19:14
【摘要】：目的結(jié)直腸癌(colorectal cancer)是消化系統(tǒng)中最常見的惡性腫瘤之一,是正常腸黏膜上皮細(xì)胞在多種致病因素的共同作用下惡性轉(zhuǎn)變的結(jié)果。目前進(jìn)展期結(jié)直腸癌患者5年生存率僅有30%,其低生存率的問題亟待解決。結(jié)直腸癌復(fù)發(fā)轉(zhuǎn)移正是當(dāng)前結(jié)直腸癌低生存率主要原因。因此,深入理解結(jié)直腸癌發(fā)生轉(zhuǎn)移的機制是解決結(jié)直腸癌患者低生存率的關(guān)鍵。隨著腫瘤研究的深入,以DNA甲基化為重要組分的表觀遺傳學(xué)修飾在結(jié)直腸癌發(fā)生發(fā)展過程中發(fā)揮的作用逐步得到重視。研究報道,DNA甲基化發(fā)生于結(jié)直腸癌早期,甚至早于遺傳學(xué)改變,在早期診斷篩查結(jié)直腸癌方面具有重要的臨床應(yīng)用價值。由此,研究結(jié)直腸癌早期發(fā)生的異常甲基化基因修飾中,是否存在能夠預(yù)測結(jié)直腸癌轉(zhuǎn)移或者患者生存率的風(fēng)險因素,通過干預(yù)基因甲基化水平,是否其對癌細(xì)胞遷移運動能力產(chǎn)生影響,有助于深入結(jié)直腸癌復(fù)發(fā)轉(zhuǎn)移的發(fā)病機制,實時監(jiān)測結(jié)直腸癌發(fā)展動態(tài),同時為結(jié)直腸癌治療新策略提供理論基礎(chǔ)。因此,本研究主要分析結(jié)直腸癌發(fā)生過程中異常的DNA甲基化修飾,同時分析其與結(jié)直腸癌轉(zhuǎn)移的關(guān)系。方法1.應(yīng)用Illumina humanmethylaiton 450K芯片(HM450K)檢測分析結(jié)直腸癌與癌旁組織間差異甲基化基因;利用DAVID在線網(wǎng)站(https://david.ncifcrf.gov/)注釋分析差異甲基化基因的生物信息學(xué)功能,如信號通路、生物學(xué)功能等�；贖M450K芯片結(jié)果,我們發(fā)現(xiàn)細(xì)胞黏附信號通路(cellular adhesion moleculars,CAMs)中緊密連接蛋白-11(CLDN11)在結(jié)直腸癌組織明顯高甲基化,由此我們將研究CLDN11甲基化在結(jié)直腸癌發(fā)生發(fā)展過程中發(fā)揮的作用;2.隨后我們應(yīng)用熒光定量甲基化特異性聚合酶鏈?zhǔn)椒磻?yīng)(quantitative methylation specific polymerase chain reaction,qMSP)在125對結(jié)直腸癌組織及癌旁組織中檢測CLDN11甲基化水平;并結(jié)合患者臨床病理信息,分析CLDN11甲基化與臨床特征相關(guān)性;3.應(yīng)用雙熒光素酶報告基因?qū)嶒?分析CLDN11啟動子片段是否具有啟動基因表達(dá)功能;4.應(yīng)用熒光定量逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(quantitative reverse transcriptionpolymerase chain reaction,qRT-PCR)檢測100對結(jié)直腸癌組織及對應(yīng)的癌旁組織中CLDN11 m RNA表達(dá)水平,分析CLDN11甲基化和表達(dá)相關(guān)性;5.應(yīng)用qMSP和qRT-PCR檢測正常腸黏膜上皮細(xì)胞株(NCM460)和4株結(jié)直腸癌細(xì)胞株(HCT116,COLO205,SW620和HT29)中CLDN11甲基化和表達(dá)水平情況;6.配置不同濃度5-aza-dC去甲基化藥物改變結(jié)直腸癌細(xì)胞CLDN11甲基化水平,應(yīng)用qMSP和qRT-PCR觀察CLDN11甲基化和表達(dá)變化,確定最佳藥物濃度;7.應(yīng)用Transwell遷移實驗,觀察CLDN11甲基化改變對結(jié)直腸癌細(xì)胞株遷移能力的影響;8.通過公共腫瘤數(shù)據(jù)庫TCGA(https://cancergenome.nih.gov/),整合CLDN11甲基化數(shù)據(jù)、表達(dá)數(shù)據(jù)和患者臨床病理信息,大樣本驗證CLDN11甲基化與表達(dá)相關(guān)性及與結(jié)直腸癌患者的無復(fù)發(fā)生存期相關(guān)性;結(jié)果1.根據(jù)HM450K芯片結(jié)果,共篩選出差異甲基化位點4047個,覆蓋1725個參考基因。基于HM450K芯片注釋分析結(jié)果,我們發(fā)現(xiàn)細(xì)胞黏附信號通路(cellular adhesion moleculars,CAMs)中CLDN11在結(jié)直腸癌組織明顯高甲基化;2.與癌旁組織相比,結(jié)直腸癌組織中CLDN11甲基化率顯著增高(P=3.71E-23)。結(jié)合患者臨床數(shù)據(jù)分析后發(fā)現(xiàn),發(fā)生淋巴結(jié)轉(zhuǎn)移結(jié)直腸癌患者組織中,CLDN11甲基化率高于未發(fā)生淋巴結(jié)轉(zhuǎn)移的結(jié)直腸癌患者(P=0.01);3.雙熒光素酶報告基因?qū)嶒灠l(fā)現(xiàn),插入的被檢的CLDN11啟動子區(qū)域片段具有啟動表達(dá)功能(P=0.002);4.q RT-PCR結(jié)果顯示,相較與癌旁組織,CLDN11 m RNA表達(dá)水平在結(jié)直腸癌組織中顯著降低(P=0.002);并且CLDN11甲基化與表達(dá)呈負(fù)性相關(guān)(r=-0.19,P=0.04),提示CLDN11啟動子甲基化是其表達(dá)降低的原因之一;5.在我們培養(yǎng)的4株結(jié)直腸癌細(xì)胞中,HCT116、COLO205和HT29結(jié)直腸癌細(xì)胞株中CLDN11的甲基化率高于NCM460,而SW620中甲基化率明顯低于NCM460;同時我們檢測細(xì)胞中CLDN11 mRNA表達(dá)水平,結(jié)果發(fā)現(xiàn)HCT116、COLO205和HT29結(jié)直腸癌細(xì)胞株中CLDN11 mRNA表達(dá)水平低于NCM460,而SW620細(xì)胞中CLDN11 mRNA表達(dá)水平高于NCM460;該結(jié)果同樣提示CLDN11啟動子甲基化是其表達(dá)降低的原因之一;6.基于結(jié)直腸癌細(xì)胞中CLDN11甲基化和表達(dá)情況,我們最后選擇HCT116細(xì)胞株進(jìn)行5-aza-dC去甲基化藥物處理。qMSP和qRT-PCR實驗結(jié)果發(fā)現(xiàn),使用9μmol/L 5-aza-dC處理HCT116時,CLDN11甲基化率改變最顯著;7.Transwell實驗證明結(jié)直腸癌細(xì)胞株降低CLDN11甲基化水平后,細(xì)胞遷移能力明顯減弱;8.基于TCGA甲基化和表達(dá)數(shù)據(jù)結(jié)果證實,結(jié)直腸癌組織中CLDN11甲基化程度與其表達(dá)成負(fù)性相關(guān)(r=-0.21,P=0.000035);結(jié)合患者臨床數(shù)據(jù)顯示,CLDN11高甲基化狀態(tài)與結(jié)直腸癌低無復(fù)發(fā)生存期(relaspe free survival,RFS)相關(guān)(Log rank P=0.04)。結(jié)論經(jīng)過實驗證實CLDN11甲基化參與結(jié)直腸癌轉(zhuǎn)移。并且結(jié)合公共數(shù)據(jù)庫大樣本數(shù)據(jù)分析顯示,CLDN11甲基化是預(yù)示結(jié)直腸癌復(fù)發(fā)潛在的預(yù)后評估分子標(biāo)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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
本文編號：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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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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