【作者】 蔡国响；

【导师】 师英强； 蔡三军； 施达仁； 孙孟红； 管祖庆；

【作者基本信息】 复旦大学 ， 肿瘤学， 2006， 博士

【摘要】 目的：研究散发性大肠癌的抑癌基因启动子甲基化和临床病理学特征以及遗传学改变（微卫星不稳定、染色体不稳定以及相关癌基因、抑癌基因表达）的关系，探讨表遗传学检测的临床意义以及表遗传学机制和遗传学机制之间的相互关系。方法：对71例散发性大肠癌（SCRC）采用甲基化特异性PCR的方法行p14^ARF、hMLH1、p16^INK4a、MGMT和MINT1共5个基因启动子甲基化的检测，选择BAT25和BAT26两个位点进行微卫星不稳定（MSI）检测、进行流式细胞术检测分析倍体类型以及进行p14^ARF、hMLH1、p16^INK4a、MGMT、k-ras、APE、p53、BAX和TGFβRⅡ共9个基因的免疫组化检测。分析p14^ARF、hMLH1、p16^INK4a、MGMT和MINT1基因启动子甲基化以及CpG岛甲基子表型（CIMP）和SCRC临床病理特征、MSI、染色体不稳定（CIN）以及相关基因表达的关系。结果：第一部分：1、p14^ARF、hMLH1、p16^INK4a、MGMT和MINT1基因启动子甲基化的阳性率分别为28.2％（20／71）、9.9％（7／71）、25.4％（18／71）、36.6％（26／71）和49.3％（35／71），CIMP阳性率为21.1％（15／71）；2、hMLH1基因启动子甲基化（+）的SERC具有右半结肠癌多发（P=0.018）和低分化癌多见（P=0.032）的特征。p16^INK4a基因启动子甲基化（+）的SCRC具有结肠癌多发（P=0.043）、低分化癌多见（P=0.000）、淋巴结转移多见（P=0.034）、分期较晚（P=0.046）的特征。p14^ARF、MGMT和MINT1三个基因的启动子甲基化均和各项临床病理学指标无显著性关系；3、CIMP（+）SCRC中右半结肠癌（40％vs12.5％，P=0.024）、低分化癌（46.7％vs14.3％，P=0.012）、淋巴结转移（86.7％vs48.2％，P=0.008）、TNMⅢ／Ⅳ期（86.7％vs50.0％，P=0.011）所占比例均显著高于CIMP（—）者。第二部分：1、71例患者共检出二倍体18例，异倍体50例（3例因CV值大于8％从分析中剔除）：2、MSI的阳性率为9.9％（7／71），所有阳性者均为BAT25和BAT26同时阳性；3、除了MGMT外，p14^ARF（P=0.062）、hMLH1（P=0.074）、p16^INK4a（P=0.053）和MINT1（P=0.002）基因启动子甲基化均表现为二倍体多发的倾向。但是仅MINT1基因启动子甲基化和二倍体的关系达到统计学显著性意义。CIMP（+）者也表现为二倍体多发的显著性特点（P=0.003）；4、hMLH1（P=0.001）和MINT1（P=0.055）基因启动子甲基化（+）者具有MSI多见的倾向。p14^ARF、p16^INK4a、MGMT基因启动子甲基化和MSI无显著性关系。CIMP（+）者中MSI的阳性率大于CIMP（—）者（20.0％vs7.1％），但P值为0.158，未达到统计学显著性意义；5、微卫星和染色体稳定型（微卫星稳定且为二倍体，MACS型）SCRC的hMLH1基因启动子甲基化的阳性率显著低于MSI型（MSI，无论二倍体或异倍体）（7.7％vs57.1％，P=0.031），MINT1基因启动子甲基化的阳性率显著高于CIN型（微卫星稳定且为异倍体）（69.2％vs35.4％，P=0.029），p16^INK4a基因启动子甲基化阳性率似高于CIN型（46.2％vs16.7％，P=0.057）。MACS型的CIMP阳性率显著高于CIN型（46.2％vs8.3％，P=0.004），而MACS型和MSI型在CIMP阳性率方面无显著性差异。第三部分：1、p14^APF、hMLH1、p16^INK4a、MGMT、k-ras、APC、p53、BAX和TGFβRⅡ蛋白阳性表达率分别为68.6％（48／70）、76.8％（53／69）、61.8％（42／68）、87.3％（62／71）、43.7％（31／71）、42.3％（30／71）、47.9％（34／71）、71.4％（50／70）和59.2％（42／71）：2、hMLH1（P=0.046）、MGMT（P=0.010）基因的启动子甲基化和相应基因的失表达密切相关，而p14^ARF和p16^INK4a基因启动子甲基化和相应基因的失表达无显著性关系；3、MINT1基因启动子甲基化和突变型p53蛋白表达密切相关（P=0.024），MINT1基因启动子甲基化（+）者突变型p53蛋白阳性表达率低于MINT1基因启动子甲基化（—）者；4、MGMT基因启动子甲基化（+）者具有k-ras蛋白高表达的趋势（P=0.070）。CIMP和APC、p53、BAX、TGFβRⅡ蛋白表达均没有显著性关系，但和k-ras蛋白表达显著相关（P=0.043），CIMP（+）者k-ras蛋白阳性表达率显著高于CIMP（—）者。结论：第一部分：hMLH1、p16^INK4a基因启动子甲基化和CIMP具有显著的临床病理学意义。hMLH1基因启动子甲基化（+）的SCRC具有右半结肠癌多发和低分化癌多见的显著性特征。p16^INK4a基因启动子甲基化（+）的SCRC具有结肠癌多发、低分化癌多见、淋巴结转移多见和分期较晚的显著性特征。CIMP（+）的SCRC具有右半结肠癌多发、低分化癌多见、常有淋巴结转移和分期较晚的显著性特点。第二部分：1、MINT1、hMLH1基因启动子甲基化以及CIMP和基因组遗传学不稳定性（CIN和MSI）具有密切的关系。MINT1基因启动子甲基化（+）者及CIMP（+）者均表现为二倍体多发的显著性特点，而hMLH1基因启动子甲基化（+）者则具有MSI多见的显著性特点；2、MACS型的SCRC是SCRC的一个独立亚型，具有和CIN型及MSI型不同的独特的临床病理和基因表达特征。MACS型SCRC的hMLH1基因启动子甲基化的阳性率显著低于MSI型，MINT1基因启动子甲基化的阳性率显著高于CIN型，CIMP阳性率显著高于CIN型。提示多基因同时甲基化可能为MACS型SCRC的重要发病机制。hMLH1基因启动子甲基化可能为MSI型SCRC的特异性发病机制，而在MACS型SCRC的发生发展过程中可能并不扮演重要角色；3、CIN、MSI和多基因启动子甲基化（CIMP）三者之间反映了表遗传学机制和遗传学机制既相互竞争又相互依存的复杂关系。CIN机制是相对独立于MSI机制及表遗传学机制（多基因启动子甲基化）之外的。MSI机制和表遗传学机制可能是一种相互依存的关系，但不是完全重叠的关系，相当一部分的MSI发生在表遗传学机制的基础上，但也有部分的MSI并没有显著的表遗传学背景。第三部分：1、hMLH1、MGMT基因启动子甲基化和相应基因的失表达密切相关；2、MINT1基因启动子甲基化（+）者呈现突变型p53蛋白低表达的显著性特点；3、MGMT基因启动子甲基化（+）者具有k-ras蛋白高表达的倾向（P=0.070）。而这种关系可能是通过MGMT蛋白的失活导致k-ras基因突变引起的；3、CIMP和k-ras蛋白的激活表达密切相关。这种关系可能是通过MGMT基因启动子甲基化导致MGMT蛋白失表达继而引起k-ras基因突变的途径。更多还原

【Abstract】 Objectives: To explore the correlation between the promotor hypermethylation of suppressor genes and clinicopathological features as well as genetic changes including microsatellite instability（MSI）, chromosomal instability（CIN） and the expression of several oncogenes and suppressor genes in sporadic colorectal cancers（SCRC）.Methods: Detecting the promotor hypermethylation of five genes including p14^ARF, hMLH1, p16^INK4a, MGMT and MINT1 with methylation specific PCR, evaluating the microsatellite instability status with two microsatellite loci of BAT25 and BAT26, analyzing the ploidy with flow cytometry and detecting the protein expression of nine genes (p14^ARF, hMLH1, p16^INK4a, MGMT, k-ras, APC, p53, BAX and TGF β R II) among 71 SCRC patients and exploring the correlation between genes promotor hypermethylation and clinicopathological characteristics as well as the genetic mechanism of SCRCs.Results: Part 1: The positive rates of genes promotor hypermethylation of p14^ARF, hMLH1, p16^INK4a, MGMT , MINT1 and CIMP were 28. 2%（20/71）, 9. 9%（7/71）, 25. 4%（18/71）, 36. 6%（26/71）, 49. 3%（35/71）and 21.1%（15/71） respectively. SCRCs with hMLH1 gene promotor hypermethylation were more likely to happen to the right hernicolon（P — 0.018） and to be poorly-differentiated adenocarcinomas（P=0.032）. p16^1NK4a gene promotor hypermethylation was significantly correlated with the proneness of colonic location （P=0.043）, poor differentiation （P=0.000）, lymph node metastasis（P=0.034）and later stages （P=0.046）. The proportion of right-sided colonic cancers （40 %vs12.5%, p=0.24）, poorly-differentiated cancers（46.7%vs14.3%, P=0.012）, cancers with lymph node metastasis （86.7%vs48.2%, P=0.008） and cancers with TNM III/IV stage （86.7%vs50.0%, P=0.011） in SCRCs showing CIMP was significantly higher than that of those without CIMP.Part 2: 18 and 50 cases showed diploidy and aneuploidy respectively among 71 patients. 3 cases with CV value higher than 8 percent were excluded from the ploidy analysis. The positive rate of MSI among SCRC patients was 9. 9 percent （7/71）. p14^ARF（P=0.062）, hMLH1 （P=0.074）, p16^INK4a（P=0.053） and MINT1 （P=0.002） gene promotor hypermethylation and positive CIMP（P=0.003） displayed the inclination of diploidy. SCRCs with hMLH1 （P=0.001） or MINT1 （P=0.055） gene promotor hypermethylation were significantly more likely to be MSI. The positive rate of MSI in positive CIMP patients was higher than that of negative CIMP ones in the present study, but the difference was not statistically significant（P=0.158）. CIMP was significantly more frequent in microsatellite and chromosomal stable （MACS） SCRCs （diploid and microsatellite stable）than that in CIN phenotype （aneuploid and microsatellite stable） （46.2%vs8.3%,P=0.004）. The hMLH1 promotor hypermethylation was less prevalent in MACS SCRCs than that in MSI phenotype （MSI regardless of the ploidy） （7.7%vs57.1 %, P=0.031）. Both the MINT1 （69.2%vs35.4%,P=0.029） and the p16^INK4a（46.2%vs16.7%, P=0.057） gene promotor hypermethylation were more common in MACS SCRCs than that in CIN phenotype. Part 3: The positive rates of protein expression of p14^ARF, hMLH1, p16^INK4a, MGMT, k-ras, APC, p53, BAX and TGF β R II were 68. 6% （48/70）, 76. 8% （53/69）, 61. 8% （42/68）, 87. 3% （62/71）, 43. 7% （31/71）, 42. 3% （30/71）, 47.9% （34/71）, 71.4% （50/70） and 59. 2% （42/71） respectively. Promotor hypermethylation of hMLH1 （P=0.046） or MGMT （P=0.010） gene was remarkably associated with the loss of protein. Promotor hypermethylation of MINT1 gene was significantly correlated with the accumulation of mutant-type p53 protein（P=0.024）. There was a tendency of activated k-ras protein expression in SCRCs with MGMT gene promotor hypermethylation（P=0.070） or positive CIMP（P=0.043） compared with the counterpart.Conclusions: Part 1: SCRCs with hMLH1 gene promotor hypermethlation have an obvious inclination to locate on the right semicolon and to be poorly-differentiated. SCRCs with p16^INK4a gene promotor hypermethlation are more likely to be colonic cancers, to bepoorly-differentiated, to metastasize to lymph nodes and to be in the later stages. SCRCs with positive CIMP are significantly inclined to occur to the right hemicolon, to have poor differentiation, to have lymph node metastasis and to be in the later stages. Part 2: There was an intimate relationship between epigenetic changes and genetic instability. Cancers with MINT1 gene promotor hypermethylation or positive CIMP are significantly more likely to be diploid while cancers with hMLH1 gene promotor hypermethylation are significantly inclined to demonstrate MSI. MACS SCRCs may compose a unique phenotype with distinct clinicopathological characteristics and gene expression profile from CIN and MSI phenotype. MACS SCRCs have a significantly lower rate of hMLH1 gene promotor hypermethylation than MSI phenotype and a remarkable higher rate of MINT1 gene promotor hypermethylation or CIMP than CIN phenotype, indicating that MACS SCRCs may develop along a pathway characterized by CIMP shared by MSI phenotype but hMLH1 gene promotor hypermethylation may not play an important role in the carcinogenesis of MACS SCRCs. The correlation among CIN, MSI and CIMP may be complicated, which reflects the competent and dependent relationship between epigenetic and genetic mechanisms. CIN mechanism may be independent of MSI and epigenetic mechanisms. A considerable part of SCRCs with MSI, but not all, may develop from a background of epigenetic instability. Part3: Promotor hypermethylation of hMLH1 or MGMT gene may cause the loss of protein. SCRCs with MINT1 gene promotor hypermethylation has a significant lower rate of accumulated mutant-type p53 protein, while those with MGMT gene promotor hypermethylation（P=0.070） or positive CIMP（P=0.043） have a relatively higher rate of activated k-ras protein expression compared with the counterpart. That the loss of MGMT protein can cause k-ras gene mutation may explain the close relation among CIMP, MGMT gene promotor hypermethylation and k-ras protein expression.更多还原