【作者】 张华；

【导师】 府伟灵；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2006， 博士

【摘要】 目的1.建立稳定的、可检测出目的片段中全部CpG位点甲基化状态的重亚硫酸盐测序技术。2.采用重亚硫酸盐测序技术,绘制散发性结直肠癌患者hMLH1基因启动子区CpG岛甲基化可变位点（Methylation Variable positions,MVPs）图谱。3.统计学分析MVPs图谱与患者年龄、性别、肿瘤发生部位、浸润程度、分化程度、淋巴结转移有无、临床病理分期和hMLH1蛋白表达的相关性,进一步阐明其表观遗传学异常机制。方法1.重亚硫酸盐测序方法的建立1.1正常人p16基因的甲基化检测选择多种肿瘤表现为CpG岛甲基化的p16基因为模型。目的序列为p16基因组5′端启动子区19556-20355的800bp片段（GenBank ID:AF 527803）。由Methprimer软件分析可知:目的片段含有两个CpG岛,共有46个CpG位点;同时由Methprimer软件设计出与重亚硫酸盐修饰后序列完全互补且不含任何CpG位点的引物:5′-GTAGGTGGGGAGGAGTTTAGTTT-3′（sense,23bp）;5′-AACTCCTCATTC CTC TTCCTTAACT-3′（anti-sense,25bp）。扩增片段位于靶序列的19632至20343位置,大小为712bp。收集正常人EDTA-K₂抗凝外周血,采用EZNA^? Blood DNA Kit提取全血基因组DNA,并用紫外分光光度计定量。采用EZ DNA Methylation Kit^TM对全血基因组DNA进行重亚硫酸盐修饰。以修饰后基因组DNA为模板,采用TaKaRa Ex Taq^TM Hot StartVersion Polymerase进行PCR扩增反应,得到长度为712 bp的目的片段。用核酸外切酶Ⅰ（ExonucleaseⅠ,ExonⅠ）和虾碱性磷酸酶（Shrimp Alkaline Phosphatase,SAP）对PCR产物进行纯化。采用测序试剂盒-BigDye V3.1 kit进行测序PCR反应,然后用ABI 3100型测序仪直接测序。1.2肿瘤细胞株甲基化检测从液氮中取出冻存的肿瘤细胞株SW480,使之复苏,以含10%小牛血清5A-medium培养液,置37℃,5%CO₂孵箱内培养。每天观察细胞的生长,收集对数生长期细胞,调整细胞数为5×10⁵。提取肿瘤细胞基因组DNA,进行重亚硫酸盐修饰后,PCR扩增出目的片段,直接测序（方法同前）。2.结直肠癌中hMLH1基因启动子区CpG岛MVPs图谱的绘制与分析2.1目的基因的分析及引物设计检索DNA甲基化数据MethDB,同时,参考文献和NCBI OMIM数据库,选择与结直肠癌发生表观遗传学异常机制相关的基因-hMLH1为研究对象。在NCBI的Gene数据库检索,获取其参考序列（GenBankID:NM-000249）。将获取的参考序列递交DBTSS数据库,查找转录起始点,确定其启动子区域,以该区域作为我们研究的目的片段。将hMLH目的片段递交至MethPrimer,经Methprimer软件分析可知:目的片段含有两个CpG岛,CpG岛1（CGⅠ-Ⅰ,324 bp,1280-1603）和CpG岛2（CGⅠ-Ⅱ,167 bp,1693-1859）共有46个CpG位点。由Methprimer软件设计出靶向两个CpG岛的重亚硫酸盐测序引物:5′-AGGATTTTTTGTTTTGTGATATTTG-3′（sense,25bp）和5′-TTAACCCTACTCTTATAACCTCCC-3′（anti-sense,24bp）,扩增片段为493 bp（1127-1619）;5′-GGGAGGTTA TAAGAGTAGGGTTAA-3′（sense,24bp）和5′-AAAATACCTTCAACCAATCACC-3′(（anti-sense,22bp）,扩增片段为423bp（1596-2018）。2.2远端正常组织甲基化检测提取组织DNA,采用重亚硫酸盐测序方法检测其甲基化状况。2.3肿瘤组织甲基化检测采用克隆测序来检测其甲基化状态。2.3.1肿瘤细胞的挑选-手工显微切割由于肿瘤组织成分比较复杂,除了肿瘤细胞外,还混有非肿瘤细胞成分,为避免这些细胞对肿瘤细胞甲基化结果的判定,故应将之尽量除去。将冻存标本行OTC包埋,-20℃条件下,切5μm厚冰冻切片一张,苏木素染色,置显微镜下观察:肿瘤细胞呈巢状排列,细胞核深染、较大且形状不规则;正常粘膜上皮排列整齐、细胞核无异形性;富含血管和炎性细胞的间质成分呈弥散分布,细胞核无异形性;平滑肌细胞多呈束状排列,细胞核为杆状,形状规则。因此,根据细胞的排列特点和细胞核形态,区分出肿瘤细胞和非肿瘤细胞,用手术刀尽量去掉非肿瘤细胞,再用切片机切20μm厚冰冻切片3～5张,置入盛有200μl PBS缓冲液的1.5ml离心管中。重复上述操作3～5次,最后收集20片左右的组织,进行后续DNA提取。2.3.2克隆测序2.3.2.1 T-A克隆首先制备大肠杆菌感受态细胞,然后用乙醇法进行目的PCR产物的纯化,再将纯化后的PCR产物与载体pMD 18-T Vector进行连接,再进行大肠杆菌的高效转化。2.3.2.2通用引物验证阳性克隆经过前期测序试验,我们发现:甲基化修饰片段以反向测序效果好,正向测序信号衰减快。因此,克隆测序必须保证方向与反向一致,故需选出正向插入的克隆,排除反向插入的克隆。所以,我们采用通用引物来验证阳性克隆:用M13R和目的片段的正向引物进行PCR扩增,就可选出正向插入的克隆,而排除反向插入的克隆。2.3.2.3测序每个样本均随机挑选20个单菌落,通过通用引物验证后,选出10个正向插入的阳性克隆,用通用引物-M13R进行测序。2.3.2.4 MVPs图谱的绘制采用BIQ_Analyzer软件分析30例样本共600个克隆的CpG位点甲基化分布和频率,从而绘制hMLH1基因启动子区CpG岛MVPs图谱。3.肿瘤组织hMLH1蛋白表达的检测4%多聚甲醛固定的标本进行石蜡包埋,制成5μm的石蜡切片,进行HE染色和免疫组化染色。显微镜观察,根据阳性细胞数和染色强度判定结果。4.MVPs图谱的分析统计学分析MVPs图谱与患者年龄、性别、肿瘤发生部位、浸润程度、分化程度、淋巴结转移有无、临床病理分期和hMLH1蛋白表达的相关性。结果1.重亚硫酸盐测序方法的建立以p16基因为模型,成功地扩增出712bp的目的片段,建立了稳定的重亚硫酸盐测序技术平台-可检测出目的片段中全部CpG位点的甲基化状况。测序结果显示:所有非甲基化的胞嘧啶均转化为胸腺嘧啶,而甲基化的胞嘧啶保持不变,说明重亚硫酸盐修饰完全。测序图谱清晰、波峰适中、无杂带。测序结果显示:正常人目的片段中的46个CpG位点均为非甲基化位点;SW480细胞株目的片段中的46个CpG位点均为甲基化位点。2.结直肠癌中hMLH1基因启动子区CpG岛MVPs图谱的绘制2.1远端正常组织甲基化检测以修饰后DNA为模板,成功地扩增出长度为493bp和423bp的目的条带。测序结果显示:目的片段中的46个CpG位点均为非甲基化位点。2.2肿瘤组织甲基化检测采用克隆测序的方法,检测了结直肠癌患者肿瘤组织hMLH1基因甲基化状况。结果:30例样本中,共有10例为甲基化阳性。其中,有6例的甲基化集中在CGⅠ-Ⅰ的第1至第28个CpG位点,其CGⅠ-Ⅱ所有CpG位点均为非甲基化;有4例为CGⅠ-Ⅰ非甲基化,但CGⅠ-Ⅱ为甲基化,其甲基化集中在CGⅠ-Ⅱ的第6和第7个CpG位点。2.3 hMLH1基因启动子区CpG岛MVPs图谱的绘制在分析远端正常组织甲基化和肿瘤组织甲基化结果的基础上,得到以CpG岛位点为横标、甲基化百分率为纵坐标的MVPs图谱。3.肿瘤组织MLH1蛋白表达的检测30例散发性结直肠癌（sporadic colorectal carcinoma,SCRC）患者的肿瘤组织中:15例hMLH1表达为阳性,占50%。其中,有4例为阳性、11例为弱阳性。另15例hMLH1表达为阴性。4.hMLH1基因启动子区CpG岛MVPs图谱的分析4.1 hMLH1基因启动子区CpG岛甲基化与蛋白表达的关系4.1.1患者hMLH1基因启动子区CpG岛甲基化与蛋白表达的关系30例SCRC患者中:6例CGⅠ-Ⅰ甲基化患者肿瘤组织的蛋白表达阴性率为100%（6/6）,24例CGⅠ-Ⅰ非甲基化患者肿瘤组织的蛋白表达阴性率为37.5%（9/24）。经x²检验,CGⅠ-Ⅰ甲基化与hMLH1蛋白表达缺失存在显著性相关（P＜0.05）。4例CGⅠ-Ⅱ甲基化患者肿瘤组织的蛋白表达阴性率为0%（0/4）,26例CGⅠ-Ⅱ非甲基化患者肿瘤组织的蛋白表达阴性率为57.7%（15/26）。经x²检验,CGⅠ-Ⅱ甲基化与hMLH1蛋白表达缺失无显著性相关（P＞0.05）。4.1.2 CGⅠ-Ⅰ中关键CpG位点的查找为了找到影响hMLH1蛋白表达的关键位点,进一步分析了6例蛋白表达阴性样本中CGⅠ-Ⅰ所有34个CpG位点的甲基化状况。采用BiQ_Analyzer软件计算出所有34个CpG位点的甲基化百分率。甲基化率为41～60%（High区）的CpG位点有:4,2,9,10,1,3,6,7,8,11,12,13,14,15,17,23,21,5,16;甲基化率为21～40%（Middle区）的CpG位点有:22,18,19,27;甲基化率为1～20%（Low区）的CpG位点有:25,26,28,24,20;甲基化率为0%（None区）的CpG位点有:29,30,31,32,33,34（注:按甲基化率的从高至低排序）。此外,通过与Herman所设计的引物的比较,发现CGⅠ-Ⅰ中:4,2,9,10,1,3等位点甲基化频率高于Herman的引物包含的CpG位点。这一结果提示在MVPs图谱的基础之上,能帮助研究者更好地选择研究CGIs甲基化状态和模式的位点。4.2 CGⅠ-Ⅰ甲基化的临床意义经x²检验,在结直肠癌患者年龄、性别、肿瘤发生部位、淋巴结转移情况、Duck’s分期、肿瘤浸润程度方面,CGⅠ-Ⅰ甲基化阳性率无显著性差异（P＞0.05）;但在肿瘤分化程度方面,阳性率有显著性差异（P＜0.05）,提示CGⅠ-Ⅰ甲基化的检测还可作为判断结直肠癌自然病程和预后的参考指标。结论1.以p16基因为模型,建立了稳定的重亚硫酸盐测序技术平台-可检测出目的片段中全部CpG位点的甲基化状况,为下一步甲基化可变位点（MVPs）图谱的绘制奠定了良好的基础。2.分析30例样本的hMLH基因启动子区甲基化结果,发现:CGⅠ-Ⅰ甲基化与hMLH1蛋白表达缺失有显著性相关,而CGⅠ-Ⅱ甲基化与hMLH1蛋白表达缺失无显著性相关,提示CGⅠ-Ⅰ甲基化可能导致hMLH1蛋白不表达。而在CGⅠ-Ⅰ的34个CpG位点中,1至28为甲基化,29至34为非甲基化。故CGⅠ-Ⅰ的1至28 CpG位点可能为导致hMLH1蛋白表达缺失的关键区域。根据其甲基化阳性率,可将这34个CpG位点分为4组:非甲基化组（甲基化率为0%）、低频率组（甲基化率为1～20%）、中频率组（甲基化率为21～40%）和高频率组（甲基化率为41～60%）。这一结果提示在MVPs图谱的基础之上,能帮助研究者更好地选择研究CpG岛甲基化状态和模式的位点。3.在恶性度极高的低分化散发性结直肠癌,其CGⅠ-Ⅰ甲基化率显著高于高中分化者,提示CGⅠ-Ⅰ甲基化的检测还可作为判断散发性结直肠癌自然病程和预后的参考指标。更多还原

【Abstract】 BackgroundsCpG islands（CGIs） of human genome,located mainly in the promoter as well as the first extron regions,are GC rich short stretches（100-1000bp） and they link with～60%of human coding genes.Methylation of CpG sites in the promoter regions of genes can lead to decreased expression and silencing of the tumor suppressor genes and contribute to oncogenesis.However,the transcriptional silencing of affected genes can occur only when the abnormal methylation occurs in specific CpG sites.MVPs（Methylation variable positions） mean the distribution and incidence of abnormal methylation CpG sites in different diseases.It is a precise analysis for quantitative evaluation of global CGIs methylation status and patterns at genomic levels.Methylation of CpG sites in the promoter regions of hMLH1 gene can decrease its mRNA level or protein expression,affect its normal repairing function,lead to delayed correction of cancer related genes mutation and contribute to colorectal cancer.Previous researchers have studied methylation of the hMLH1 promoter using MSP method,but this method can only analyze CpG sites in the PCR primers.To characterize precisely the regions involved in the epigenetic silencing of hMLH1,we collected colonic samples from sporadic colorectal carcinoma patients,conducted the NaHSO₃ treatment-sequencing method for methylation analysis of hMLH1 promoter,and explored the correlation between our findings and clinical indicators.Objectives1.To construct an effective and stable method for quantifying all the CpG sites of target sequence by bisulfite sequencing.2.Map the complete methylation status of the hMLH1 promoter-a region that contains 46 CpG sites in 30 sporadic colorectal carcinomas patients. 3.Detect the expression of hMLH1 by immunohistochemistry.4.Analysis on the relationship between MVPs of hMLH1 promoter and the expression of hMLH1 together with clinical significance.Methods1.Construction of method by bisulfite sequencing1.1 Detection on methylation of the p16 promoter 5’—CpG of healthy peopleThe p16 promoter region from19556 to 20355（GenBank ID:AF 527803）was selected as the target sequence.Primers were designed by Methprimer software.The primers were completely complement to the bisulfite modification sequence and did not contain any CpG sites.Primers used were 5’-GTAGGTGGGGAGGAGTTTAGTTT-3’（sense） and 5’-AACTCCTC ATTCCTCTTCCTTAACT-3’（anti- sense）,which were used to amplify a 712 bp product.Total genomic DNA was isolated from anticoagulated whole blood of healthy people, and modified by sodium Bisulfite.And then,the modified DNA was amplified by PCR, followed by ExonⅠand SAP purification.Subsequently,the PCR products were analyzed by automated DNA sequencing（ABI）.1.2 Detection of methylation of the p16 promoter 5’-CpG of tumor cell line-SW480The tumor cell line-SW480 was cultured in 5A-medium supplemented with 10%CS at 37℃in 5%CO₂ Total genomic DNA was isolated from the SW480 cells.And then, methylation of p16 promoter 5’-CpG was detected as described previously.2.Mapping the complete methylation status of hMLH1 promoterAccording to Gene database of NCBI,some refereces and NCBI OMIM database,we obtain the reference sequence（GenBank ID:NM-000249）and present it to DBTSS database to find transcription initiation site and determine promoter region and take this region as our target fragment.Then the hMLH fragment was presented to Methprimer to prove it containing two CGIs was selected as the target sequence.Two pairs of primers were designed by Methprimer software. Primers were:5’-AGGATTTTTTGTTTTGTGATATTTG-3’（sense） and 5’-TTAA CCCTACTCTTATAACCTCCC-3’（anti-sense）;5’-GGGAGGTTATAAGAGTAGGGT TAA-3’ （sense） and 5’-AAAATACCTTCAACCAATCACC-3’（anti-sense）,which could amplify 493 bp and 423 bp products,respectively.Thirty sporadic colorectal carcino-mas were obtained from surgical patients and this set of samples has been character-rized previously for clinicopathological parameters. 2.1 Detection of methylation of hMLH1 promoter in normal colorectal tissueAll of these 30 patients corresponding non-cancer colorectal tissue was obtained from a tumour-free location,which was at least 2 cm far away from the tumor and which was confirmed to be without any tumor cell infiltration by histology.Total genomic DNA was isolated from the 30 samples.And then,Genomic DNA of all samples was analyzed by the bisulfite genomic sequencing technique after bisulfite conversion as reported previously.2.2 Detection of methylation of hMLH1 promoter of cancerous samples2.2.1 Treatment of cancerous samplesIsolate cancerous cells under a light microscope followed hematoxylin staining,wash several times in phosphate-buffered saline（PBS） to remove cell debris.Extract Genomic DNA of these cancerous cells and treat them with sodium bisulfite.And then,the modified DNA was amplified by PCR.2.2.2 Cloning and sequencingThe PCR products were purified by alcohol and cloned into TA vector.Then insertpositive clones were selected by PCR.Sense primers used were:5’-AGGATTTTTTGTTTTG TGATATTTG-3’ and 5’-GGGAGGTTATAAGAGTAGGGTTAA-3’.Anti-sense primer used was:5’-CAGGAAACAGCTATGAC-3’.They were taken to amplify 539 bp and 469 bp products,respectively.Ten clones per samples were sequenced using the ABI sequencing system.3.Detecting the expression of hMLH1 proteinParaffinized serial sections were cut at 5μm for immunohistochemistry and HE staining.Immunostaining was performed with antibodies directly against the C-terminal region of human MLH1 protein.Results1.Method of bisulfite sequencing was constructed successfully.2.The hypermethylation of hMLH1 gene was not detected in normal tissues.But it was detected in tumor tissues:the hypermethylation of CGIs 1 was in 20%（6/30） patients and CGIs 2 was 13.33%（4/30）.3.The hMLH1 protein was detected in 50%tumor tissues.There was a highly significant correlation between the hypermethylation of CGIs 1 and loss of hMLH1 expression.In CGIs 1,CpG positions from 1 to 28 were hypermethylated. 4.There was no significant correlation between the hypermethylation of CGIs 1 and Patients age,gender,tumor location,invasive depth,lymphnode metastasis,and Dukes stage.But,there was a highly significant correlation between the hypermethylation of CGIs 1 and poorly differentiated carcinoma.Conclusions1.The construction of bisulfite sequencing should be helpful to further analysis on global CGIs methylation status of tumor suppressor genes.2.In CGIs 1,CpG positions from 1 to 28 are the critical region which could influence expression of hMLH1.3.MVPs of hMLH1 promoter could be used as a promising prognostic indicator of human sporadic colorectal carcinoma.更多还原