Wnt拮抗基因SFRP1、SFRP2启动子区甲基化与贲门腺癌关系的研究

目的研究贲门腺癌（GCA）中分泌型卷曲相关蛋白1（SFRP1）、分泌型卷曲相关蛋白2（SFRP2）基因启动子区甲基化状态与贲门腺癌的关系。方法采用甲基化特异性PCR（MSP）方法检测GCA癌组织及相应癌旁正常组织中SFRP1和SFRP2基因的甲基化状态,分析其与临床病理特征之间的关系。结果94例GCA组织中SFRP1、SFRP2基因甲基化率分别为87．2％（82／94）和83．0％（78／94）,显著高于癌旁正常组织14．9％（7／47）和553％（26／47）（P〈0．001）。GCA患者中无淋巴结转移组SFRP1基因甲基化发生率73．7％（28／38）显著低于有淋巴结转移组的甲基化率96．4％（54／56）（P〈0．05）;SFRP2基因的甲基化与有无淋巴结转移无关。SFRP1和SFRP2基因的甲基化与GCA的组织分化无关。63例贲门腺癌组织中SFRP1和SFRP2基因同时发生甲基化,其中有淋巴结转移的36例高于无淋巴结转移的27例,高中分化腺癌组26例,低于低分化腺癌组37例,侵及肌层及浆膜层的16例低于侵及周围软组织的47例,但以上差异均无统计学意义（P〉0．05）。结论SFRP1、SFRP2基因可能参与了贲门腺癌的发生发展,并且SFRP1高甲基化状态与贲门腺癌的恶性行为有关。     

Nm23 expression in human gastric cancers - possible correlation of nm23 with lymph-node metastasis

In order to elucidate the relationship between nm23 and metastasis in human gastric cancers, we analyzed gene and protein expression of nm23-H1 using Northern blot and immunohistochemical techniques. nm23-H1 gene expression was identified in 17 out of 19 gastric cancer tissues. The signals in the tumor tissues presenting regional lymph node metastasis seem to be lower than those in the tumor tissues without regional lymph node metastasis, suggesting a role of nm23-H1 in the regional lymph node metastasis in the gastric cancers. However, the protein expression detected immunohistochemically was not correlated to the gene expression, partly because of difficulty in quantifying the amount of protein. Expression of the nm23-H1 gene as well as the nm23-H1 protein in the tumor tissues was higher than those in the corresponding normal mucosae. This suggests a linkage of nm23-H1 in the process of the gastric cancer progression. We also analyzed the sequence abnormalities of the nm23-H1 gene in the gastric cancer tissues using a direct sequencing technique, no mutations were observed.     

MicroRNA-335的表遗传学调控机制及其与胃癌患者临床病理特征的关系

目的:探索miR-335基因启动子区甲基化状态对胃癌组织及细胞系中miR-335表达水平的影响,以及miR-335基因启动子区甲基化状态与胃癌患者临床病理特征以及预后的关系。方法:收集2012年7月1日-2014年7月1日中国医科大学附属第四医院就诊经病理诊断为原发性胃癌并行根治性手术的108例新鲜胃癌组织及配对癌旁组织,1株永生化的胃黏膜上皮细胞系(GES-1)和4株胃癌细胞系(SGC-7901、MKN-45、BGC-823和AGS)。实时荧光定量PCR(qRT-PCR)检测胃癌细胞株及108例胃癌患者肿瘤组织中miR-335的表达水平。甲基化特异性PCR(MSP)方法检测胃癌细胞系及胃癌患者肿瘤组织中miR-335的基因启动子区甲基化状态。分析miR-335基因启动子区甲基化状态对胃癌患者临床病理特征的影响。结果:qRT-PCR检测结果显示,miR-335在胃癌细胞株中的表达水平显著低于正常胃黏膜上皮细胞株GES-1［MKN-45,0.154±0.016-fold(P＜0.01);SGC-7901,0.138±0.013-fold(P＜0.01);BGC-823,0.432±0.076-fold(P＜0.01);AGS,0.749±0.072-fold(P=0.01)］。miR-335在108例胃癌组织中的表达水平较癌旁组织存在明显下降,差异显著（P＜0.001）。MSP的实验结果表明,MKN-45、SGC-7901、AGS和BGC-823细胞株均存在基因启动子区异常高甲基化状态。miR-335基因启动子区的高甲基化状态与肿瘤大小(P=0.004)、淋巴结转移(P=0.046)、淋巴管浸润(P=0.001) 和miR-335 低表达 (P<0.001) 显著相关。结论:miR-335启动子区的高甲基化状态抑制了miR-335在胃癌细胞中的表达,miR-335的基因启动子区异常高甲基化状态与胃癌患者的肿瘤大小、淋巴结转移以及淋巴管浸润显著相关。     

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.     

MSP法检测胃癌组织中RASSF1A基因启动子区甲基化改变

目的：探讨抑癌基因RASSF1A启动子区CpG岛甲基化与胃癌及临床病理特征的关系。方法：采用甲基化特异性PCR（methylation—specific PCR,MSP）法检测60例胃癌组织及相应癌旁组织和30例对照组织中RASSF1A基因启动子区甲基化状态。结果：胃癌组织中RASSF1A基因启动子区CpG岛甲基化率为65．0％（39／60）,艋著高于癌旁组织6．7％（4／60）,及对照组0％（0／30）（P〈0．01）。胃癌组织中不同年龄、性别、分化程度及淋巴结转移与否的RASSF1A基因甲基化率的差异均无统计学意义。结论：胃癌中RASSF1A基因启动子区的高甲基化提示其与胃癌的发生密切相关,MSP法对RASSF1A基因启动子区甲基化的检测有望成为胃癌早期监测的重要方法。     

胃癌组织及相关淋巴结中端粒酶逆转录酶基因启动子区域甲基化检测的临床意义

[目的]检测胃癌患者肿瘤组织及其相应的癌旁组织和淋巴结组织中端粒酶逆转录酶(hTERT)基因启动子区域甲基化状态,并探讨其甲基化状态的改变与临床病理特征的关系.[方法]运用甲基化特异性PCR(MSP)方法,检测52例手术切除胃癌组织、癌旁组织及相关淋巴结中hTERT基因启动子区域甲基化状念、以同一标本正常组织作为阴性对照.[结果]正常胃黏膜组织未检测出hTERT表达、胃癌组织及癌旁组织、转移淋巴结中均检测出hTERT表达.转移淋巴结、胃癌组织中hTERT基因的甲基化阳性率分别为81.6％(31/38)、71.1％(37/52),明显高于癌旁组织的29.5％(13/52)(P＜0.01).胃癌组织hTERT基因甲基化阳性率与胃癌的临床分期、组织分化程度、肿瘤大小有相关性(P＜0.05).癌旁组织hTERT基因甲基化阳性率和胃癌的临床分期、肿瘤大小、组织分化程度、淋巴结转移具有相关性(P＜0.05).转移淋巴结hTERT基因 甲基化阳性率则与临床及病理特征无关.[结论]胃癌组织及转移淋巴结中存在hTERT基因启动子区域的异常甲基化调控、可能参与了胃癌的发生与发展.     

Promoter CpG methylation of tumor suppressor genes in colorectal cancer and its relationship to clinical features

Aberrant promoter methylation of CpG islands of tumor suppressor genes inhibits expression of the genes and may lead to tumorigenesis. We investigated the aberrant methylation profile of potential tumor suppressor genes of p15, p16, SOCS-1, and Wnt signaling pathway in colorectal cancers and correlated the data with clinical findings. Cancerous and nearby non-cancerous tissues of 185 sporadic colorectal cancer samples were studied. Methylation specific PCR was performed to explore the mechanism of inactivation in p15, p16, SOCS-1, E-cadherin, APC, GSK-3beta, and Axin1 genes. Aberrant promoter methylation in p15, p16, SOCS-1, E-cadherin, APC, GSK-3beta, and Axin1 genes were 5.9, 7.0, 3.8, 5.9, 12.4, 2.2, and 0% for cancerous tissues, respectively, whereas the frequencies were 3.8, 0, 0, 7.0, 2.7, 0.5, and 0% for nearby non-cancerous tissues, respectively. The frequency of aberrant promoter methylation of cancerous tissues was significant higher than non-cancerous tissues in p16, SOCS-1, and APC genes (p<0.05) and methylation status of these genes had no clear relationship with clinical parameters. Of the 66 patients who showed at least one aberrant promoter methylation in the tumor-suppressor genes, 5 (7.6%) patients demonstrated multiple methylation phenotype (methylation > or =3) and associated with increased lymph node metastasis (p=0.036). Our findings suggest that inactivation of some tumor suppressor genes through aberrant promoter methylation of CpG islands may play a role in the development of colorectal cancer and methylation inactivation of these genes except p16 and SOCS1 may occur at the precancerous stage. Multiple methylation pathways may be involved in the tumorigenesis of colorectal cancer and associated with aggressiveness of clinical disease.     

Promotor hypermethylation of p14ARF is a key alteration for progression of oral squamous cell carcinoma

We investigated the promotor hypermethylation status of multiple genes in 49 oral squamous cell carcinomas (OSCC), using the methylation-specific PCR (MSP) assay. The genes examined included p16INK4a, p14ARF, RB1, p21Waf1, p27Kip1, PTEN, p73, 0(6)-MGMT, and GST-P. Detailed clinicopathological data, such as patient age, sex, tobacco use, alcohol consumption, lesion site, degree of tumor differentiation, tumor size, presence of lymph node metastasis, and clinical stage, were collected for all 49 samples. Overall, gene methylation was detected in 46.9% (23/49) of samples and was closely correlated with tobacco use or/and alcohol consumption. Of the genes investigated, p16INK4a, p14ARF, 0(6)-MGMT, RB1, PTEN, and p27Kip1 were found to be methylated in 34.7%, 20.4%, 12.2%, 10.2%, 6.1%, and 4.1% of these 49 tumors, respectively, but methylation of p21Waf1, p73, and GST-P was not detected at all. Methylation frequencies were much higher for each gene when computed among informative cases only. Concurrent promotor hypermethylation of p16INK4a and p14ARF correlated significantly with tumor size, lymph node metastasis, and stage III/IV advanced OSCC; p14ARF hypermethylation, in particular, was significantly associated with both lymph node metastasis and late clinical stage. Our results suggest that DNA methylation of multiple genes, especially hypermethylation of the p14ARF promoter, is common in OSCC and is associated with the use of tobacco and/or alcohol consumption. For this type of cancer, the data further implicates gene methylation as playing an important role in tumor progression.     

Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line

The interleukin-mediated Janus kinase (JAK)/STAT pathway plays a crucial role in carcinogenesis. Recently, increased STAT3 activity was found in hepatocellular carcinoma and multiple myeloma in which there was silencing of SOCS-1 (suppressor of cytokine signalling-1) by gene promoter hypermethylation. We investigated the expression level of interleukin-6 (IL-6) and SOCS-1 in gastric cancer cell lines. Expression of SOCS-1 correlated with IL-6 level in most of the cell lines, except for AGS cells in which SOCS-1 was absent despite a high level of IL-6 production. Methylation analysis by methylation-specific polymerase chain reaction and bisulphite sequencing revealed that CpG island of SOCS-1 was densely methylated in AGS cells. Demethylation treatment by 5'aza-deoxycytidine restored SOCS-1 expression and also suppressed constitutive STAT3 phosphorylation in AGS cells. Moreover, methylation of SOCS-1 was detected in 27.5% (11 of 40) of primary gastric tumours samples, 10% (one of 10) of adjacent noncancer tissues but not in any (zero of nine) normal gastric mucosa. Methylation of SOCS-1 also correlated with the loss of mRNA expression in some primary gastric cancers. In conclusion, this is the first report to demonstrate that hypermethylation of SOCS-1 led to gene silencing in gastric cancer cell line and primary tumour samples. Downregulation of SOCS-1 cooperates with IL-6 in the activation of JAK/STAT pathway in gastric cancer.     

Promoter methylation of cyclin D2 gene in gastric carcinoma

Methylation of CpG island in the promoter has been recognized as an important mechanism for regulation of gene expression. Although considerable work has been done on the epigenetic control of tumor suppressor genes, little is known about the potential role of promoter CpG demethylation in the activation of oncogenes. The cyclin D2 gene is overexpressed in a subset of gastric carcinoma. To determine whether hypomethylation of cyclin D2 is involved in stomach carcinogenesis, we studied methylation of CpG islands in the cyclin D2 gene by methylation-specific PCR in 34 gastric carcinoma specimens, 21 corresponding non-neoplastic mucosae, and 8 gastric carcinoma cell lines. We also measured levels of cyclin D2 mRNA in 23 of the gastric carcinoma cases and in the gastric carcinoma cell lines. Hypomethylation of the cyclin D2 promoter was found in 24 (71%) of the 34 tumor tissues and in 6 (29%) of the 21 corresponding non-neoplastic mucosa, the incidence being significantly different (p=0.002; Fisher's exact test). Moreover, hypomethylation of cyclin D2 was more common in stage III and IV tumors than in stage I and II tumors (p=00.014; Fisher's exact test). All of three cell lines with promoter hypomethylation expressed detectable levels of cyclin D2 mRNA. Treatment of cyclin D2-negative cells lines harboring promoter hypermethylation with demethylating agent, 5-Aza-2'-deoxycytidine, led to a reactivation of cyclin D2 expression. These results suggest that DNA hypomethylation is a mechanism underlying the increased expression of cyclin D2 in cancer cells and that demethylation of cyclin D2 may be involved in development and progression of gastric carcinoma.     

Clinicopathological variables associated with lymph node metastasis in submucosal invasive gastric cancer

Background We aimed to elucidate clinicopathological variables associated with lymph node metastasis of submucosal invasive gastric cancer. Methods Specimens were surgically resected from 201 patients who had primary submucosal gastric cancer. We studied 39 consecutive patients with lymph node metastasis and 162 patients without lymph node metastasis. We compared the following clinicopathological characteristics of the patients in relation to lymph node metastasis: age, sex, tumor size, histology, extent of submucosal invasion, lymphatic and venous invasion, and ulceration of the tumor. Submucosal invasion was divided subjectively into sm1, sm2, and sm3 (representing invasion of the upper-, middle-, and lower-third of the submucosa, respectively). We also studied the relationship between lymph node metastasis of submucosal gastric cancer and immunohistochemistry for p53, Ki67, vascular endothelial growth factor (VEGF), α-fetoprotein, sLe^a, and dendritic cells (DCs). Results In terms of conventional pathological factors, lymph node metastasis in submucosal gastric cancer was related to tumor size (P = 0.002), depth of submucosal invasion (P = 0.001), lymphatic invasion (P < 0.0001), and venous invasion (P = 0.012). Lymph node metastasis in sm1 gastric cancer was significantly related to VEGF expression (P = 0.047). Also, lymph node metastasis in sm3 gastric cancer was significantly correlated with DC expression (P = 0.016). Multivariate analysis showed that tumor size, tumor invasion depth in the submucosal layer, and lymphatic invasion were independent predictors of nodal metastasis in submucosal gastric cancer. Conclusion Conventional pathological factors, such as tumor size, depth of submucosal invasion, and lymphatic invasion, have a significant influence on lymph node metastasis. VEGF expression and DC expression may be helpful predictors of lymph node metastasis in patients with sm1 and sm3 gastric cancer, respectively.     

APE表达水平与胃癌临床病理因素的相关性分析

目的：研究胃癌患者不同胃黏膜组织无嘌呤无嘧啶核酸内切酶（apurinic/apyrimidinic endonuclase APE）的表达水平与临床病理因素的相关性.方法：利用已构建好的包含208例胃癌患者胃癌、正常胃黏膜和转移淋巴结的组织芯片,用免疫组化方法检测不同组织APE表达水平,结合患者的临床资料进行统计分析.结果：正常组织和转移淋巴结中细胞核、细胞质APE表达水平与临床各病理因素之间没有明确相关性;胃癌组织中胞核表达与肿瘤浸润深度（P=0.000）、有无淋巴结转移（P=0.010）、TNM分期（P=0.000）有明显相关,浸润深度越深、出现淋巴结转移和TNM分期越晚,则胞核表达越弱,胞核表达还显示出与性别有一定相关性,男性表达水平较女性高（P=0.048）,胞质表达水平则与性别无明确相关性,只显示与淋巴结转移（P=0.017）和TNM分期（P=0.019）有关,有淋巴结转移和TNM分期较晚的患者胞质表达水平较弱.结论：随着胃癌的进展,肿瘤分期越晚、浸润深度越深和出现淋巴结转移,则胃癌组织中胞质和胞核的APE表达水平越低.     

FHIT和p16基因甲基化与胃癌的关系研究

目的探讨FHIT和p16基因甲基化在胃癌发生发展中的作用。方法应用甲基化特异性PCR（MSP）,检测胃癌组织和正常胃黏膜组织中FHIT基因和p16基因的启动子CpG岛的甲基化状态。结果胃癌组织中FHIT和p16基因甲基化阳性率分别为51.4%和56.8%,两者的甲基化均与胃癌患者年龄、性别、Lauren分型、Borrmann分型、淋巴结转移以及TNM分期无关（P〉0.05）,且两者的甲基化不存在明显正相关关系（P〉0.05,γ=0.17）。结论 FHIT基因和p16基因的甲基化修饰在胃癌发生发展中均起重要作用,两者可能是胃癌发生的早期事件。     

EphA1基因在结直肠癌中的表达及临床意义

目的：探讨EphA1在结直肠癌中的表达及其临床意义。方法:利用荧光实时定量RT-PCR检测EphA1 在肠癌细胞系和组织中的表达,并分析甲基化状态。免疫组织化学染色分析EphA1蛋白在肠癌组织中的表达及其临床意义。结果:EphA1在肠癌细胞中不同程度表达,并存在甲基化。EphA1 mRNA在癌旁正常黏膜和肿瘤组织中表达差异无统计学意义（P=0.496）。EphA1蛋白在肠腺瘤中弥漫强阳性,在癌组织中主要为低或缺失表达,低表达多见于低分化腺癌(P＝0.027)、伴有更深的肠壁浸润深度（P=0.002)和淋巴结转移患者（P=0.019）。EphA1下调多见于中晚期病例（P=0.002),下调较上调患者预后不良。结论:EphA1在结直肠癌的发生发展中可能具有一定肿瘤抑制作用,有可能成为预测结直肠癌恶性程度和预后的新指标。     

食管鳞癌中DACT2基因表达及甲基化状态研究

[目的]检测食管鳞状细胞癌(ESCC)中DACT2基因表达及启动子区甲基化状态,探讨DACT2基因在食管鳞癌发生发展中的作用.[方法]分别应用逆转录—聚合酶链反应(RTPCR)以及甲基化特异性PCR(MSP)的方法检测DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-aza-dC)处理前后的食管癌细胞系(TE1、TE13、T.Tn、Eca109)以及食管鳞癌组织及相应癌旁组织中DACT2 mRNA表达情况及启动子区甲基化状态.[结果]经5-aza-dC处理后4种食管癌细胞系中DACT2基因的表达均增高.4种未经5-aza-dC处理的食管癌细胞系中DACT2基因呈高甲基化状态.应用5-aza-dC处理后,DACT2基因在4种细胞系中均呈非甲基化状态.DACT2基因在食管鳞癌组织中的表达显著低于癌旁组织(0.66±0.53 vs 0.95±0.64,t=-2.43,P=0.018),并与淋巴结转移密切相关(t=-2.030,P=0.048).食管鳞癌组织中DACT2基因的启动子区甲基化率显著高于癌旁组织(50.0％ vs 21.1％,x2=9.439,P=0.002),并与TNM分期、组织学分化程度和淋巴结转移密切相关(P均＜0.05).发生DACT2基因甲基化的食管鳞癌组织中DACT2基因的表达量显著低于未发生甲基化的食管鳞癌组织(0.46±0.32 vs 0.78±0.61,t=-2.341,P=0.023).[结论]DA CT2基因在食管鳞癌中的异常低表达与食管鳞癌的发生、发展密切相关,且其启动子区甲基化可能是导致其表达沉默的机制之一.     

miR-3666在胃癌中的表达及其临床意义

目的:检测胃癌组织中miR-3666的表达并分析其与临床病理特征的关系。方法:采用茎环逆转录实时定量PCR技术检测80例胃癌手术标本与相对应的癌旁组织中miR-3666的相对表达量,并分析其与胃癌临床病理参数和预后的关系。结果:miR-3666在胃癌组织中表达与癌旁组织相比显著下调（P<0.001）,miR-3666的表达与胃癌淋巴结转移（P=0.002）和TNM分期（P=0.017）相关,与年龄、性别、浸润深度、肿瘤位置及分化程度无关（P>0.05）。合并淋巴结转移的胃癌组织中miR-3666的表达低于无淋巴结转移的胃癌组织（P<0.001）。miR-3666低表达与高表达的患者1年及3年生存率分别为78.0%、22.6%和96.9%、48.1%,中位生存率分别为16个月和29个月,差异有统计学意义（P<0.001）。COX预后分析发现miR-3666低表达和肿瘤TNM分期是胃癌患者预后的独立影响因素。结论:miR-3666在胃癌组织中的表达量明显降低,提示miR-3666可能参与了胃癌的发生发展,并且与胃癌的淋巴结转移相关;miR-3666低表达提示胃癌患者预后不良。     

钙粘蛋白基因高甲基化与胃癌发生、发展的关系

背景与目的:有研究报道基因启动子区CpG岛甲基化引起的钙粘蛋白(E-cadherin,E-cad)基因失活可能在胃癌的发生发展中起重要作用.本研究拟通过检测胃癌组织、癌前病变组织和正常对照组织中E-cad基因启动子区CpG岛甲基化水平及其表达,结合临床病理资料,分析其与胃癌发生、发展的关系.方法:用甲基化特异性聚合酶链反应(methylafion-specific PCR,MSP)检测41例胃癌组织、40例癌前病变组织和38例正常对照组织中E-cad基因启动子5'CpC岛甲基化.将PCB扩增产物克隆并测序.应用免疫组化检测基因的蛋白表达.结果:胃癌组织中E-cad基因甲基化阳性率为19.5%(8/41),癌前病变组织为2.5%(1/40),而正常对照组织为0.0%(0/38),前组与后两组之间的差异均有统计学意义(P＜0.05).胃癌组织中E-cad蛋白阳性率为70.7%(29/41),癌前病变组织为97.5%(39/40),正常对照组织为100.0%(38/38),前组与后两组之间的差异均有统计学意义(P＜0.05).低分化型胃癌组织的E-cad基因甲基化阳性率明显高于高分化型(43.8% vs.4.0%)(P＜0.05).有淋巴结转移的胃癌组织中E-cad基因甲基化阳性率与无转移组的差异有统计学意义(33.3% vs.5.0%)(P＜0.05).浸润深达浆膜层的胃癌组织中甲基化阳性率与未达浆膜层组的差异有统计学意义(35.0% vs.4.8%)(P＜0.05).胃癌组织中,E-cad基因甲基化阳性组的蛋白阳性率显著低于甲基化阴性组(0.0% vs.87.9%)(P＜0.05).结论:胃癌组织中存在E-cad基因启动子5'CpG岛高甲基化;从胃癌前病变、低度恶性到高度恶性的胃癌,E-cad基因的高甲基化水平呈不断增加的趋势.