抑癌基因启动子甲基化和蛋白表达与胃癌临床病理学特征的关系及其对胃癌生物学功能的影响

发布时间：2018-03-25 09:19

本文选题：胃癌　切入点：抑癌基因　出处：《浙江大学》2016年博士论文

【摘要】：研究背景胃癌的发病率和死亡率高居全球恶性肿瘤的第4和第3位,为我国最常见的恶性肿瘤之一。近年来研究表明,胃癌的发生发展是一个多因素、多阶段、多基因异常表达累积的病变过程。除经典的基因缺失与突变机制外,表观遗传学的改变在胃癌的发生发展过程中逐渐受到重视,其中基因启动子异常甲基化被认为是调控基因表达的第三种重要机制。肿瘤抑癌基因(tumor suppressor gene,TSG)是生物体内细胞增殖、分化和凋亡过程中的负性调节因子之一。由抑癌基因启动子高甲基化引起的基因失活有助于细胞的无限制生长,促进肿瘤的形成。因此,对这方面进行研究有助于解释细胞增殖、分化和癌变等过程。抑癌基因异常甲基化常在肿瘤早期被检测出来,通过筛选胃癌相关的基因甲基化谱,能为肿瘤的早期诊断、治疗和预后提供理论及实验依据。研究目的通过分析胃癌组织中抑癌基因p16、hMLH1和CDH1启动子区甲基化状态及其蛋白表达水平,明确两者之间的相关性;统计分析这3个基因启动子区甲基化状态和蛋白表达水平与临床病理参数、预后参数之间的关系,揭示这3个基因在胃癌发生发展中的作用;在胃癌细胞系和正常胃黏膜上皮细胞系中过表达抑癌基因hMLH1,通过细胞功能实验,初步探讨hMLH1在胃癌中的抑癌作用机制。研究方法1、检测胃癌组织中p16、hMLH1和CDH1基因启动子区甲基化及其与临床病理特征和预后的关系随机选择经病理确诊的120例原发性胃癌患者,所有患者术前均未行放疗和化疗,收集胃癌根治术后肿瘤组织及其相距5cm的配对癌旁组织。用QIAampDNA Mini Kit试剂盒提取DNA,继以亚硫酸氢盐转化。采用甲基化特异性PCR(methylation-specific PCR,MSP)方法检测上述样本中 p16、hMLH1 和 CDH1 基因启动子CpG岛甲基化的状况,在SPSS 18.0统计软件中用卡方检验或Fisher精确检验分析每个基因甲基化程度与临床病理参数之间的关系。探讨p16、hMLH1和CDH1基因甲基化状态与患者总体生存期的关系用Kaplan-Meier法。整体比较采用Log-rank检验并绘制生存曲线。2、检测胃癌组织中p16、hMLH1和E-cadherin蛋白表达水平及其与临床病理特征和预后的关系对120对胃癌组织和癌旁组织进行常规苏木精-伊红(Hematoxylin-eosin staining,HE)染色和免疫组织化学(Immunohistochemistry,IHC)染色。所有步骤依据产品说明书进行,利用PBS(0.01mol/L,pH7.4)替代I抗作为阴性对照。免疫组化染色结果判定:p16和E-cadherin蛋白免疫染色阳性信号定位于细胞浆,hMLH1蛋白免疫染色阳性信号定位于细胞核,呈棕黄色颗粒,每个标本观察2个有代表性的高倍视野,每个视野计数100个细胞,确定阳性细胞数所占的百分比并取它们的平均值,阳性细胞数≤20%为阴性(-),阳性细胞数20%为阳性(+)。在SPSS 18.0统计软件中用卡方检验或Fisher精确检验分析每个蛋白表达与临床病理学参数之间的关系。探讨p16、hMLH1和E-cadherin蛋白表达水平与患者总体生存期的关系用Kaplan-Meier法。整体比较采用Log-rank检验并绘制生存曲线。用Spearman相关性分析探讨基因启动子甲基化状态与其蛋白表达之间的关系。3、胃癌细胞系和正常胃黏膜细胞系中hMLH1基因启动子区甲基化和mRNA表达水平检测体外培养胃癌细胞系(MKN-74、MKN-45、SGC-7901、HGC-27、MGC-803、BGC-823和AGS)和正常胃黏膜上皮细胞系GES-1。细胞接种于含10ml/L胎牛血清(56℃灭活30min)及DMEM/F12培养基中,在37℃、含5%CO2的湿润空气的恒温密闭式培养箱中培养。取对数生长期的细胞数1×106进行细胞DNA抽提及重亚硫酸氢盐转化,采用MSP方法检测8种细胞株中hMLH1基因启动子甲基化状况。用Trizol试剂提取细胞总RNA,并按RT-PCR试剂盒说明书进行反转录合成cDNA;荧光定量PCR扩增,分析各细胞系中hMLH1基因mRNA的相对表达水平。获得启动区子高甲基化且mRNA表达水平下降的胃癌细胞系。4、克隆形成检测和Transwell实验构建野生型hMLH1基因慢病毒表达载体(LV-GFP-MLH1),转染启动区子高甲基化且mRNA表达水平下降的胃癌细胞系AGS,建立稳定表达外源性hMLH1和阴性对照AGS细胞株。采用克隆形成实验检测hMLH1过表达对AGS细胞克隆形成能力的影响;Transwell实验分析hMLH1过表达对AGS细胞侵袭能力的影响。研究结果1、MSP检测结果显示,120例胃癌组织中p16、hMLH1和CDH1基因启动子区甲基化阳性率分别是 36.67%(44/120)、24.17%(29/120)和 25.00%(30/120);癌旁组织中p16、hMLH1和CDH1基因启动子区甲基化阳性率分别是25.83%(31/120)、8.33%(10/120)和 14.17%(17/120)。hMLH1 和 CDH1 两个基因甲基化阳性率在胃癌组织与癌旁组织之间的差异均具有统计学意义(P0.05)。2、hMLH1基因甲基化状态与饮酒(P=0.019)、CA199水平(P0.001)和Borrman分型(P = 0.030)有关,有饮酒史患者其hMLH1甲基化率(40.00%)高于无饮酒史患者(18.89%);CA19937kU/L的胃癌患者其hMLH1甲基化率(53.57%)高于CA199≤37kU/L的胃癌患者(15.22%);Borrman Ⅰ型和Ⅱ型患者其hMLH1甲基化率(66.67%)高于Ⅲ型和Ⅳ型患者(21.93%)。CDH1基因甲基化状态与血清癌胚抗原(CEA)水平有关(P = 0.003),CEA5μg/L的胃癌患者其CDH1甲基化率(46.43%)高于CEA≤5μg/L的胃癌患者(18.48%)。hMLH1基因启动子甲基化与胃癌患者总体生存相关,是独立预后因子,hMLH1基因高甲基化胃癌患者预后更差。p16和CDH1基因启动子区甲基化各自与胃癌患者总体生存期无关,但在联合分析中,携带2个或3个基因甲基化的患者的总体预后比携带0个或1个基因甲基化的患者差(χ~2= 3.901,P = 0.048)3、免疫组化结果显示,120例胃癌组织中p16、hMLH1和E-cadherin蛋白表达阳性率分别是:36.67%(44/120)、69.17%(83/120)、68.33%(82/120);癌旁组织中,p16、hMLH1和E-cadherin蛋白表达阳性率分别是:86.67%(104/120)、94.17%(113/120)、92.50%(111/120)。p16,hMLH1 和 E-cadhein 蛋白表达阳性率在胃癌组织与癌旁组织之间的差异均具有统计学意义(P0.05)。4、胃癌p16蛋白表达水平与肿瘤大小有关(P = 0.030),肿块直径6cm患者其p16表达阳性率(44.44%)高于肿块直径≥6cm的患者(25.00%)。hMLH1蛋白表达水平与肿瘤标记物CA199(P = 0.041)、肿瘤大小(P = 0.036)有关,CA19937kU/L的胃癌患者其hMLH1蛋白表达阳性率(53.57%)低于CA199≤37kU/L几的胃癌患者(73.91%);肿块直径6cm的患者其hMLH1表达阳性率(76.39%)高于肿块直径≥6cm患者(58.33%)。E-cadherin蛋白表达情况与饮酒(P = 0.041)有关,有饮酒史胃癌患者其E-cadherin表达阳性率(53.33%)低于无饮酒史患者(73.33%)。p16、hMLH1和E-cadherin蛋白表达水平与胃癌患者总体生存期无关。胃癌组织中hMLH1基因甲基化与其蛋白表达存在负相关性(P=0.005)5、胃癌细胞系(MKN-74、MKN-45、SGC-7901、HGC-27、MGC-803、BGC-823和AGS)中hMLH1基因启动子均为甲基化,正常胃黏膜上皮细胞系GES-1中hMLH1为非甲基化。各胃癌细胞系中hMLH1的mRNA表达水平均低于正常胃黏膜上皮细胞系。将加MLH1基因过表达病毒感染的AGS细胞组标记为实验组,加阴性对照病毒感染的AGS细胞组标记为对照组。克隆形成实验显示,对照组和实验组的克隆形成数分别是180±3个和169±6个,实验组的克隆形成数高于对照组(P= 0.039)。Transwell实验结果显示,对照组和实验组的穿膜细胞数分别为135±4.08个和125±4.18个,实验组的穿膜细胞数高于对照组(P = 0.048)。结论胃癌组织中p16、hMLH1和CDH1基因启动子区异常甲基化是频发事件,多个抑癌基因启动子区异常甲基化同时存在可能是胃癌发生发展的重要机制之一。hMLH1和CDH1基因启动子区甲基化可能是胃癌癌变过程中的重要分子事件,是胃癌早期诊断的潜在分子标志物。hMLH1基因启动子区甲基化可通过下调其基因表达,促进胃癌细胞的克隆形成并增强侵袭能力,从而参与胃癌的发生发展。hMLH1启动子甲基化与胃癌患者总生存相关,是胃癌患者预后的独立危险因素;同时,多个基因甲基化也与预后相关,可为胃癌患者的精准治疗和预后预测提供理论依据。
[Abstract]:Background gastric cancer morbidity and mortality in the world's fourth and third malignant tumors, is one of the most common malignant tumor in China. Recent studies show that the occurrence and development of gastric cancer is a multi factor, multi stage, pathological process of cumulative abnormal expression of multiple genes. In addition to the gene deletion and mutation mechanism. Epigenetic changes, has attracted much attention in the development of gastric cancer, the gene promoter methylation is believed to be the third important mechanisms regulating gene expression of tumor suppressor genes (tumor, suppressor gene, TSG) is a biological cell proliferation, differentiation and apoptosis in negative regulation factor one of the unlimited growth caused by tumor suppressor gene promoter hypermethylation of gene inactivation contributes to cells, promote tumor formation. Therefore, this research helps to explain cell proliferation, Division The process and cancer. Hypermethylation of tumor suppressor gene in tumor early detection, through the screening of gastric cancer related gene methylation profiles, for the early diagnosis of cancer, and provide a theoretical and experimental basis for treatment and prognosis. The purpose of the study through the analysis in gastric cancer tissues by p16, hMLH1 and CDH1 promoter methylation and its protein expression level, correlation between; statistical analysis of the 3 gene promoter methylation status and protein expression and clinicopathological parameters, prognosis parameters, revealed that 3 genes in gastric carcinoma; expression of hMLH1 gene in human gastric cancer cells lines and normal gastric epithelial cell line, through cell function experiments, preliminary study of hMLH1 in gastric carcinoma tumor suppressor mechanism. Methods 1 detecting gastric cancer p16, hMLH1 and CDH1 gene promoter Methylation and clinicopathological characteristics and prognosis between randomly selected 120 patients with pathologically confirmed primary gastric cancer patients, all patients were not receiving preoperative radiotherapy and chemotherapy, radical resection of gastric cancer were collected after tumor tissues and paired tumor adjacent tissues. 5cm were extracted by DNA QIAampDNA Mini Kit kit, followed by bisulfite conversion. Methylation specific PCR (methylation-specific PCR MSP) method to detect the samples of p16, hMLH1 and CDH1 gene promoter CpG island methylation status, exact test to analyze the relationship between each gene methylation and clinicopathological parameters by using the chi square test or Fisher in the SPSS 18 statistical software in the study. P16, Kaplan-Meier by using the method of the relationship between hMLH1 and CDH1 gene methylation status and overall survival in patients. The Log-rank test was used to compare and draw survival curves of.2, detection of P in gastric cancer tissues 16, the expression level of hMLH1 and E-cadherin protein and its relationship with clinicopathological features and prognosis of hematoxylin and 120 pairs of gastric carcinoma and eosin (Hematoxylin-eosin staining, HE) staining and immunohistochemical staining (Immunohistochemistry, IHC). All of the steps according to the product specification, the use of PBS (0.01mol/L, pH7.4) replace I as the negative control. Immunohistochemical staining results: p16 and E-cadherin immunostaining positive signal located in the cytoplasm, hMLH1 immunostaining positive signal located in the cell nucleus, brownish yellow granules, each specimen has 2 HPF representative, each field count of 100 cells, identified the percentage of positive cells accounted for and take their average value, the number of positive cells less than 20% as negative (-), the number of positive cells was positive (+ 20%). Using the chi square statistical software in SPSS 18 Test or Fisher exact test analysis of each protein expression of relationship between parameters and clinical pathological study. P16, Kaplan-Meier by using the method of the relationship between hMLH1 and E-cadherin protein expression level and overall survival in patients. The Log-rank test was used to compare the survival curve was drawn. Analysis of the relationship between.3 gene expression and its promoter of protein by the correlation between Spearman, hMLH1 of gastric cancer cell lines and normal gastric mucosa cell line gene promoter methylation and expression of mRNA in gastric cancer cell lines cultured in vitro (MKN-74, MKN-45, SGC-7901, HGC-27, MGC-803, BGC-823 and AGS) and normal gastric epithelial cell line GES-1. cells were cultured in 10ml/L containing fetal bovine serum (56 C inactivation of 30min) and DMEM/F12 medium at 37 degrees C, 5%CO2 medium containing moist air sealed thermostatic incubator. The number of cells in logarithmic growth phase into 1 * 106 The cell extract DNA and bisulfite conversion, using MSP method to detect 8 kinds of cell line hMLH1 gene promoter methylation status. Total cellular RNA was extracted with Trizol reagent, and cDNA was synthesized by reverse transcription by RT-PCR kit; fluorescence quantitative PCR amplification, hMLH1 analysis of various cell lines relative gene mRNA the expression level of the promoter region. To obtain high methylation and low expression of mRNA in gastric cancer cell line.4, and Transwell assay to construct wild-type hMLH1 gene lentiviral vector cloning (LV-GFP-MLH1), transfection of promoter region of high methylation and low expression of mRNA in gastric cancer cell line AGS, and establish a stable expression of exogenous hMLH1 and the negative control cell line AGS. The clone forming test effect of hMLH1 overexpression on AGS cell colony formation; Transwell experimental analysis of hMLH1 over expression on invasion ability of AGS cells. Ring. Results: 1, MSP test results showed that in 120 cases of gastric cancer, p16, hMLH1 and CDH1 gene promoter methylation positive rate was 36.67% (44/120), 24.17% (29/120) and 25% (30/120); p16 in the cancer tissues, hMLH1 and CDH1 gene promoter methylation positive rate were 25.83% (31/120), 8.33% (10/120) and 14.17% (17/120) between.HMLH1 and CDH1 two gene methylation positive rate in gastric cancer tissue and paracancerous tissue were statistically significant (P0.05.2), the methylation status of hMLH1 gene and alcohol (P=0.019), CA199 (P0.001) and Borrman type (P = 0.030), drinking in patients with a history of hMLH1 methylation rate (40%) higher than that of non drinking history patients (18.89%); CA19937kU/L in gastric cancer patients with hMLH1 methylation rate (53.57%) higher than that of CA199 = 37kU/L in patients with gastric cancer (15.22%); Borrman type patients with hMLH1 methylation rate (66.67% ) higher than that of type III and type IV patients (21.93%) the methylation status of.CDH1 gene and serum carcinoembryonic antigen (CEA) level (P = 0.003), CEA5 g/L in gastric cancer patients with CDH1 methylation rate (46.43%) higher than that of CEA is less than or equal to 5 g/L (18.48%.HMLH1) in patients with gastric cancer related gene promoter Zi Jiaji the overall survival of patients with gastric cancer, is an independent prognostic factor of hMLH1 hypermethylation and worse prognosis of patients with gastric cancer.P16 CDH1 gene promoter methylation has nothing to do with their survival in gastric cancer patients in general, but in the joint analysis, the overall prognosis with 2 or 3 gene methylation in patients compared with 0 or 1 gene methylation in patients with poor (~2= 3.901, P = 0.048) 3, immunohistochemistry showed that in 120 cases of gastric cancer, p16, hMLH1 and E-cadherin protein expression positive rate respectively is: 36.67% (44/120), 69.17% (83/120), 68.33% (82/120); tumor tissues, p16 hMLH1, and E-cadheri The positive rate of N expression were 86.67% (104/120), 94.17% (113/120), 92.50% (111/120).P16, were statistically significant difference between the positive rate in gastric cancer tissue and tumor expression of hMLH1 and E-cadhein protein (P0.05).4, the expression level of p16 protein in gastric cancer is related to tumor size (P = 0.030) the diameter of tumor patients with 6cm, the positive rate of p16 (44.44%) is higher than the diameter of tumor was 6cm patients (25%) the expression level of.HMLH1 protein and tumor marker CA199 (P = 0.041), tumor size (P = 0.036), CA19937kU/L of gastric cancer patients the positive rate of hMLH1 expression (53.57%) is lower than CA199 or 37kU/L a few of the patients with gastric cancer (73.91%); the diameter of tumor patients with 6cm the positive expression rate of hMLH1 (76.39%) is higher than the diameter of the tumor more than 6cm patients (58.33%) the expression of.E-cadherin protein and alcohol (P = 0.041), a history of alcohol in gastric cancer patients with E-cadherin positive expression The rate of (53.33%) is lower than that of the non drinking history (73.33%) patients with.P16, hMLH1 and E-cadherin protein expression in patients with gastric cancer overall survival. Negative correlation between the expression of hMLH1 gene methylation and protein in gastric cancer tissues (P=0.005 5), gastric cancer cell lines (MKN-74, MKN-45, SGC-7901, HGC-27, MGC-803, BGC-823 and AGS) in the hMLH1 gene promoter was methylated hMLH1, normal gastric epithelial cell line GES-1 was unmethylated. The expression levels of hMLH1 in the gastric cancer cell line mRNA was lower than that of normal gastric epithelial cell line. The overexpression of MLH1 gene in AGS cell marker virus infection group as experimental group, and negative control AGS cells were labeled virus infection as the control group. The clone formation assay showed that the cloned control group and experimental group which were 180 + 3 and 169 + 6, the experimental group formed the number of clones was higher than the control group (P= 0.039).Transwell. The experiment results show that the number of transmembrane cells were the control group and the experimental group was 135 + 4.08 and 125 + 4.18, the number of transmembrane cells in the experimental group than in the control group (P = 0.048). Conclusion p16 in gastric cancer tissues, hMLH1 and CDH1 gene promoter methylation is a frequent event, multiple tumor suppressor gene promoter hypermethylation at the same time there may be an important mechanism of occurrence and development of gastric carcinoma.HMLH1 and CDH1 gene promoter methylation may be an important molecular event in gastric carcinogenesis, is a potential molecular marker in early diagnosis of gastric cancer by down regulating the gene expression of promoter methylation of.HMLH1 gene, promote the gastric cancer cell formation and enhance the invasion ability, and thus participate in the development of gastric cancer. The methylation of the.HMLH1 promoter associated with the overall survival of patients with gastric cancer were independent risk factors for the prognosis of gastric cancer patients; at the same time, multiple gene methylation The prognosis is also related to prognosis, which can provide a theoretical basis for accurate treatment and prognosis prediction of gastric cancer patients.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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