胃癌相关抑癌基因甲基化研究进展

DNA甲基化是一种表观遗传修饰,它与肿瘤的发生关系密切。DNA甲基化是基因表达调控的一种方式。抑癌基因启动子高甲基化可以使其表达失活,导致肿瘤发生。胃癌的发生与多基因异常表达密切相关,其中抑癌基因甲基化是胃癌发生发展的重要机制之一。     

Methylation of the hMLH1 promoter in multiple gastric carcinomas with microsatellite instability

Hypermethylation of the hMLH1 promoter is observed in the majority of sporadic gastric carcinomas with high frequency microsatellite instability (MSI), and it contributes to the genesis of MSI-positive gastric carcinoma. Multiple gastric carcinoma is known to have a higher frequency of MSI positivity than single gastric carcinoma. However, the molecular basis of MSI in these tumors remains obscure. We investigated the role of hMLH1 promoter hypermethylation in the genesis of multiple gastric carcinoma with MSI. We analyzed 33 tumors from 15 patients with multiple gastric carcinoma (12 double tumors and three triple tumors) for MSI, expression of hMLH1 and hMSH2, and hypermethylation of hMLH1 and hMSH2 promoters. High frequency MSI was found in seven out of 33 tumors (21%) in five out of 15 patients (33%). All of the tumors with high frequency MSI had a lack of hMLH1 expression, with the presence of hMSH2 expression, while all the tumors with no MSI or low frequency MSI were positive for both hMLH1 and hMSH2. All of the tumors with no expression of hMLH1 had hMLH1 hypermethylation, whereas hMLH1 hypermethylation was observed in two out of 26 (8%) tumors with no or low frequency MSI. None of the tumors showed hMSH2 hypermethylation. These results suggest that epigenetic changes in the hMLH1 promoter account for the genesis of multiple gastric carcinoma with high frequency MSI.     

hMLH1和干细胞标志物CD44在胃癌的表达及相互间的关系

目的研究错配修复基因hMLH1和干细胞标志物CD44在胃癌的表达状况、意义及相互间的关系。方法采用免疫组化SP法检测79例胃癌组织和相对应的癌旁组织中错配修复基因hMLH1和干细胞标志物CD44的表达状况,结合临床资料分析这两个指标的表达与肿瘤病理参数间的关系,运用卡方检验分析该两个指标间的相互关系。结果79例胃癌标本中,hMLH1阳性表达的有15例,占19%,,CD44阳性表达的有53例,占67.1%,两者在癌旁正常组织表达的阳性率分别为51%和15.2%,两者差异均有统计学意义(P<0.05);胃癌组织中hMLH1的表达与CD44呈负相关(P<0.05);hMLH1阴性表达、CD44阳性表达与胃癌淋巴结转移、浸润深度、TNM分期及脉管浸润呈正相关(P<0.05)。结论 hMLH1在胃癌的形成过程中起到重要作用,它与胃癌干细胞标志物CD44之间存在拮抗关系。     

RUNX3基因启动子甲基化与胃癌关系的meta分析

目的采用meta分析的方法评价RUNX3基因启动子甲基化与胃癌发生的关系。方法检索公开发表在Pubmed、EMBASE、Ovid、中国期刊全文数据库（CNKI）关于RUNX3基因启动子甲基化与胃癌发生关系的研究。应用Statall．0统计软件分析RUNX3基因启动子甲基化与胃癌发生间的关联。结果共有19项研究纳入分析．meta分析结果显示：胃癌患者癌组织中RUNX3基因启动子甲基化发生率为P=55％（95％CI：45％～66％）：胃癌患者远癌正常胃组织中RUNX3基因启动子甲基化发生率为P=18％（95％CI：7％～29％）。与远癌正常胃组织相比,胃癌组织中RUNX3基因启动子甲基化发生优势比OR=7．85（95％CI：5．81～10．61）。结论胃癌患者癌组织中RUNX3基因启动子甲基化发生率明显高于远癌正常胃组织,RUNX3基因启动子甲基化可能与胃癌的发生密切相关。     

低剂量螺旋CT扫描联合血清p16基因甲基化检测在肺癌早期诊断中的应用

目的研究在无症状的肺癌高危人群中利用低剂量CT（LDCT）联合血清p16基因甲基化检测进行肺癌早期诊断的可行性。方法肺癌高危人群入组标准：男性，年龄55～75岁；吸烟指数≥400支／年，目前仍在吸烟或戒烟不超过10年。共893例受检者被随机分为两组。一组为447例（LDCT—p16组），平均年龄66岁，进行LDCT联合血清p16基因甲基化检测；另一组为446例（CXR组），平均年龄67岁，接受后前位胸片检查。两组检查阳性病例将接受进一步组织病理学检查。并分别统计两组阳性结节检出率及肺癌检出率，并行X2检验。结果LDCT—p16组与CXR组分别有96．8％和92．8％的受检者完成了检查。LDCT—p16组中1113％病人可疑肺癌，明显高于CXR组的6．5％（P〈0．05）。其中LDCT—p16组中有7例，CXR组中有2例确诊为肺癌。LDCT—p16组肺癌检出率高于CXR组，但无统计学意义（P〉0．05）。结论低剂量CT联合血清p16基因甲基化检测是一种敏感、安全、可行的筛查早期肺癌的方法，能够取代胸片筛查早期肺癌。     

DNA甲基化和组蛋白乙酰化与胃癌的研究进展

胃癌是我国最常见的恶性肿瘤，但其具体发病机制还不是很清楚。表遗传学改变，如DNA甲基化和组蛋白修饰改变可以调节基因的表达，在肿瘤的发生和发展中可能起关键作用。近年来一些胃癌相关基因的DNA甲基化和组蛋白乙酰化改变已经得到证实，这些基因改变涉及细胞凋亡、细胞周期阻滞、分化和增殖等。     

启动子DNA甲基化调节胃癌中PDX1基因表达

目的明确PDXl启动子DNA甲基化,探讨启动子甲基化对PDXl在胃癌中表达的调节作用。方法收集3例胃癌活检组织,免疫组化检测PDXl蛋白表达：吉西他滨处理3株胃癌细胞,RT—PCR检测不同药物剂量和作用时间下PDXlmRNA表达;构建PDXl报告基因,检测启动子活性及吉西他滨处理前后启动子活性的变化;甲基化特异性PCR（MSP）检测3株胃癌细胞和8对配对胃癌组织中PDXl启动子甲基化状态。结果免疫组化结果显示胃癌中PDXl表达低于正常胃黏膜;RT—PCR显示吉西他滨使PDXlmRNA重获表达,且随剂量和时间依赖性。F383有最强启动子活性,吉西他滨显著增加了PDXl启动子活性（P〈0．05）。F383在AGS、BCG823、SGC7901中呈DNA完全甲基化状态;87．5％的胃癌组织出现F383部分甲基化,12．5％出现完全甲基化。癌旁正常组织仅有37．5％出现F383部分甲基化,未出现完全甲基化,两者比较有显著性差异（P〈0．05）。结论PDXl启动子存在DNA高甲基化,抑制了胃癌中PDXl的表达。     

Differential expression of hMLH1 in sporadic human colorectal cancer tumors and distant metastases

Somatic defects in the mismatch repair system constitute an important pathway in colorectal carcinogenesis. We have examined the expression of mismatch repair proteins in sporadic stage IV colorectal tumors and their derived metastases. Sporadic tumors were further examined for differences in expression between the tumor transition zone and the invasive front. Expression of hMSH2, hMLH1, and hPMS2 was screened immunohistochemically in 92 stage IV tumors and derived liver metastases. In cases with loss of mismatch repair protein expression, lymph node metastases were also examined. Clinicopathological parameters and Ki‐67 staining indexes were evaluated and compared. Four tumors displayed a complete loss of hMLH1/hPMS2 expression at the transition zone; however, three of these expressed both proteins at the invasive front and in liver and lymph node metastases. A further four were predominantly hMLH1/hPMS2 negative at the transition zone, but with distinct subclones of hMLH1/hPMS2‐expressing cells at the transition zone. All of these tumors expressed hMLH1/hPMS2 at the invasive front and in liver metastases, with three also expressing hMLH/hPMS2 in lymph node metastases. No significant difference in the proliferative index was observed for the hMLH1/hPMS2‐compromised group. In stage IV tumors re‐expression of hMLH1/hPMS2 occurred, leading to different patterns of expression within the primary tumor and between tumor and metastases. This may have functional importance for the chemosensitivity of metastases compared to the primary tumor.     

Fibulin-1基因启动子区域甲基化与食管癌的关系研究

目的:检测食管癌Fibulin-1基因启动子区域甲基化的情况,探讨其相关的临床意义.方法:选取黄石市中心医院2014年1月至2016年2月收治的食管鳞状细胞癌患者96例,另取同期黄石市中心医院因食管良性病变手术切除的食管组织40例作为对照.Fibulin-1基因启动子甲基化使用亚硫酸盐修饰后PCR检测.免疫组织化学检测显示Fibulin-1蛋白表达.结果:食管癌患者中Fibulin-1基因启动子甲基化阳性率为36.5％,显著高于对照组的15.0％(x2=2.517,P=0.036).免疫组织化学检测显示Fibulin-1基因启动子甲基化标本中Fibulin-1蛋白阳性表达率为90.2％.食管癌患者男女性别和不同年龄之间Fibulin-1基因启动子甲基化率无显著性差异(P＞0.05).Ⅲ,Ⅳ临床分期患者Fibulin-1基因启动子甲基化率为43.1％,显著高于Ⅰ,Ⅱ临床分期患者的28.9％(x2=2.426,P=0.046).肿瘤有淋巴结转移患者Fibulin-1基因启动子甲基化率为44.4％,显著高于无淋巴结转移患者的26.2％(x2=2.438,P=0.041).肿瘤中、低分化患者Fibulin-1基因启动子甲基化率为42.6％,显著高于高分化患者的25.7％(x2=2.431,P=0.043).结论:Fibulin-1基因启动子在食管癌组织中高甲基化,Fibulin-1基因启动子高甲基化与食管癌的临床分期、肿瘤分化程度以及有无淋巴结转移密切相关.     

错配修复基因hMLH1启动子萤光素酶报告基因载体的构建与鉴定

 目的：构建含hMLH1启动子片段的萤光素酶报告基因载体,并检测其在雌激素诱导下的转录活性。方法：以正常人基因组DNA为模板,PCR扩增hMLH1启动子片段（-1953/+53）,插入萤光素酶报告基因载体pGL3-Basic,将重组质粒转入HEK293细胞,检测在有和无雌激素诱导下萤光素酶的活性变化。结果：酶切和测序结果证实重组萤光素酶报告基因载体pGL3-Promoter1-luc构建成功。转染pGL3-Promoter1-luc后,雌激素诱导的萤光素酶活性为7.45±0.81,显著高于无水乙醇组的3.28±0.19 (n=3,P<0.001)。结论：hMLH1启动子片段存在与雌激素相关的调控序列。     

遗传性弥漫型胃癌家系上皮型钙黏素基因的分析

 摘要：目的 研究在遗传性弥漫型胃癌家系中是否存在上皮型钙黏素基因(CDH1)以及基因表达的异常。 方法 收集符合遗传性弥漫型胃癌(HDGC)诊断标准的1个家系中15例成员的外周血和组织标本。通过免疫组化和Western blot 的方法检测组织标本CDH1蛋白表达; 提取基因组DNA, 通过PCR扩增DNA直接测序检测CDH1基因16个外显子突变。用克隆测序法,鉴定CDH1基因启动子区CpG位点甲基化状况。 结果 先证者和另一胃癌患者(家系2号成员)的癌旁胃粘膜上皮细胞CDH1蛋白表达较正常胃粘膜减弱, 两者肿瘤组织的蛋白表达几乎为阴性。包括先证者在内的11例家系成员第14外显子mRNA水平2377位点存在一个C?T的单核苷酸多态性(single nucleotide polymorphism, SNP), 但未检测到16个外显子的胚系突变。相对于正常胃粘膜, 先证者和家系2号成员的胃癌组织均有CDH1基因启动子的高甲基化, 其瘤旁粘膜也有高甲基化。结论 此HDGC家系中,CDH1基因表达丢失可能和胃癌发生有关, CDH1基因外显子突变不是其肿瘤组织上皮型钙黏素蛋白表达丢失的直接原因, 基因启动子区的甲基化可能是导致其失活的原因之一。     

早期胃癌临床诊治的若干问题

胃癌是全球范围内死亡率最高的恶性肿瘤之一,而胃癌的早期发现和规范化治疗是改善胃癌患者的重要手段。随着医疗技术的发展,早期胃癌的检出率逐年提高,并且手术方式逐渐从传统开腹根治术向内镜切除和腹腔镜手术过渡。本课题组通过多年系列研究,联合国内外最新发现对早期胃癌临床生物学特征、分期系统评估和综合治疗方案等方面进行深入研究,为指导早期胃癌临床诊治提供科学依据。     

UHRF1基因在甲基化调控与血管新生中的作用

近年来大量研究证实泛素样含植物同源结构域和环指结构域1(ubiquitin-like with PHD and RING finger domains 1,UHRFl)是一种与肿瘤发生相关的核蛋白基因,在表观遗传修饰过程中具有重要的调控作用,尤其是在DNA甲基化与组蛋白甲基化的调控方面。UHRF1由于其特有的结构域,在细胞生物学功能调控中起到非常重要的作用,如细胞增殖、周期和凋亡等。另外,在多种肿瘤组织和细胞中异常高表达的UHRF1可能与肿瘤血管新生密切相关。本文主要综述UHRF1对DNA甲基化及组蛋白甲基化的调控作用,并探讨了UHRF1在血管新生过程中可能发挥的表观遗传学调控作用。     

Elevated early gastric carcinoma. Differential diagnosis as regards adenomatous polyps.

Differential diagnostic problems between gastric carcinomas and precancerous lesions with severe dysplasia have become more perceptible with the increasing number of resected early carcinomas. Although such problems come up for all macroscopic and histologic types of gastric cancer they are particularly marked between early carcinomas of the elevated type and adenomatous polyps. Elevated early carcinomas are usually highly differentiated adenocarcinomas with a morphology which often reminds of of adenomas. But sometimes the carcinomas also demonstrate convincing signs of being developed from adenomas. The criterion of distinction between intramucosal carcinomas and adenomas is invasion through the basal membrane, often difficult to evaluate. The morphological relation between elevated early gastric carcinomas and adenomas and the criterion of distinction between them were studied in 20 early gastric carcinomas of the Japanese types I and IIa, 6 intramucosal and 14 submucosal all highly differentiated adenocarcinomas, and in 42 polyps, of which 5 were of the adenomatous type. All lesions were taken from resection specimens. Among the carcinomas 5 demonstrated convincing signs of being malignant transformed adenomas. In addition, 6 carcinomas had a morphology which more or less reminded of adenomas, but their genetic origin was more uncertain. Nine carcinomas revealed no sign of an adenomatous origin. Among the 5 polyps diagnosed as adenomas 2 revealed an extraordinary degree of severe dysplasia which caused uncertainty on the benign diagnosis. The rest of the polyps were without dysplasia. The significance of invasion through the basal membrane as an indispensable factor of distinction between adenoma and carcinoma in the stomach is discussed. It is concluded that the degree of dysplasia can be so severe and the invasion so difficult to evaluate that the classification of some few tumours depends on the subjectivity of the single pathologist. Four of the tumours, 2 adenomas and 2 intramucosal carcinomas, having a remarkable macroscopic appearance like a large mucosal fold are especially mentioned. Their relation to gastric mucosal prolaps is discussed. Furthermore, a tumour apparently demonstrating only a moderate degree of dysplasia, but even so setting up metastases is mentioned in detail.     

RUNX3基因与胃癌的关系

RUNX3基因是RUNX转录因子家族成员之一，其在胃黏膜上皮生长调控、脊神经节的神经发育和T细胞分化过程中发挥重要作用。在人类多种恶性肿瘤尤其是胃癌中发现RUNX3基因表达缺失或下调。目前发现有多种机制包括杂合性缺失、高甲基化和点突变等参与了RUNX3基因在胃癌中的表达缺失或下调，其中RUNX3启动子区域CpG岛的甲基化是导致其在胃癌中失活的主要机制。随着研究的不断深入，RUNX3基因有望成为胃癌诊断的一个特异性生物学标志物和基因治疗的靶点。     

FOXA1 positively regulates gene expression by changing gene methylation status in human breast cancer MCF-7 cells

Objective: DNA methylation is an important epigenetic modification with tumor suppressor gene silencing in cancer. The mechanisms underlying DNA methylation patterns are still poorly understood. This study aims to evaluate the potential value of FOXA1 for controlling gene CpG island methylation in breast cancer. Methods: FOXA1 was down-regulated by transfection with siRNA and up-regulated by transfection with plasmid in MCF-7 cell lines. The DNA methylation and mRNA levels were examined by qMSP and qRT-PCR. The cell proliferation and apoptosis was detected by MTT and Flow cytometry. Results: Suppression of FOXA1 enhanced the methylation status of DAPK, MGMT, RASSF1A, p53, and depressed mRNA levels of these tumor suppressor genes, whereas over-expression of FOXA1 showed the opposite effects. DNMT1, DNMT3A and DNMT3B mRNA were up-regulated by siRNA knock-down of FOXA1. At the same time, FOXA1 suppression promoted cell growth and inhibited apoptosis. Conclusions: FOXA1 may be associated with methylation of the tumor suppressor genes promoter through changing DNMTs expression. FOXA1 could be a potential demethylation target for prevention and treatment of breast cancer.     

Standardized approach for microsatellite instability detection in gastric carcinomas

Microsatellite instability (MSI) defines a specific type of genetic instability. Although consensus diagnostic criteria for MSI definition in colorectal cancer have been established, their utility in other tumor types remain to be proven. Previously we developed a mathematical model for MSI definition in colorectal cancer. The aim of this study was to establish diagnostic criteria for MSI evaluation in human gastric cancer. We designed an algorithm for the efficient characterization of MSI and used it to analyze data on 7 microsatellite markers in 35 gastric carcinomas. Theoretical models considering 1, 2, or 3 populations were tested against the data collected. Also, hypermethylation of hMLH1 gene promoter and hMLH1 protein expression were studied. The observed frequencies of MSI in our series of samples best fit a 2-population model: stable and unstable, defined by instability in 2 or more of a minimum of 7 markers analyzed. MSI was observed in 29% of the tumors. Misclassification rate was <4% when any 7 loci were analyzed. MSI(+) tumors inversely associated with p53 protein overexpression. A good correlation between hMLH1 status (either protein or promoter hypermethylation) and MSI classification was observed. We have developed a simple, sensitive, and specific approach to assess the presence of MSI in gastric cancer that may have clinical applications.     

DYNC1H1 gene methylation correlates with severity of spinal muscular atrophy

Methylation profiles of CpG islands within the SLC23A2, CDK2AP1, and DYNC1H1 genes and their association with spinal muscular atrophy (SMA) severity were studied. High clinical heterogeneity of SMA suggests the existence of different factors modifying SMA phenotype with gene methylation as a plausible one. The genes picked up in our earlier genome‐wide methylation studies of SMA patients demonstrated obvious differences in their methylation patterns, thus suggesting the likely involvement of their protein products in SMA development. Significantly decreased methylation of CpG islands within exon 37 of the DYNC1H1 gene was observed in patients with a severe SMA manifestation (type I) compared to mildly affected SMA patients (types III–IV). This finding provides new information on peculiarities of methylation in clinically different types of SMA patients and gives a clue for identification of new SMA modifiers.