EB病毒阳性和阴性胃癌中视网膜母细胞瘤基因甲基化状态及其表达

目的:了解EB病毒(Epstein-Barr virus,EBV)相关胃癌(EBVaGC)和阴性胃癌(EBVnGC)组织中视网膜母细胞瘤(Rb)基因甲基化状态及蛋白表达,探讨EBV感染与Rb基因甲基化的关系.方法:采用甲基化特异性PCR(MSP)对各种临床病理指标匹配的23例EBVaGC和25例EBVnGC组织及相应癌旁组织中Rb基因启动子区域的甲基化状态进行检测,并采用免疫组化技术检测两种胃癌组织中Rb蛋白的表达.结果:胃癌与相应癌旁组织中Rb基因启动子区的甲基化率分别为64.6%(31/48)和39.6%(19/48),差异有显著性(P<0.05);EBVaGC组织中Rb基因启动子区的甲基化率为82.6%(19/23),高于EBVnGC中的检出率(48.0%,12/25),差异有显著性(P<0.05);EBVaGC和EBVnGC组织中Rb蛋白的表达率分别为52.2%(12/23)和72.0%(18/25),差异无显著性(P□0.05);胃癌组织中Rb启动子基因甲基化与蛋白表达无明显负相关.结论:Rb异常甲基化在胃癌细胞中较常见,EBV可以诱导Rb基因甲基化,影响其基因表达而参与EBVaGC的发生.     

CAGE 基因mRNA水平表达与直肠癌关系的初步研究

目的:探讨直肠癌组织中癌相关基因(cancer-associated gene,CAGE)mRNA水平表达与直肠癌发生及临床分期、淋巴结转移的关系。方法:对我院2014年2月-2014年10月间肠镜室检查的标本,按病理检查结果分为3组:直肠癌组(80例;为直肠癌患者标本),大肠腺瘤组(38例;为大肠腺瘤患者标本),对照组(40例;为距肿瘤边缘10 cm以上标本)。采用RT-PCR法检测并比较各组CAGE在mRNA水平表达情况。甲基化特性PCR法(methylation-specific PCR,MSP)检测CAGE基因启动子的甲基化情况。将直肠癌患者分为CAGE基因甲基化患者组和CAGE基因去甲基化患者组,比较两组临床分期和淋巴结转移率。结果:与对照组相比,直肠癌组和大肠腺瘤组的CAGE mRNA水平表达明显升高(P0.01);直肠癌组高于大肠腺瘤组(P0.01)。3组间的基因启动子区的去甲基化阳性率差异有统计学意义(P0.01)。在直肠癌组中,CAGE去甲基化阳性患者的淋巴结转移率为73.8%(48/65),CAGE去甲基化阴性患者的淋巴结转移率为33.3%(5/15),两者相比差异有统计学意义(P0.01)。临床分期为I-Ⅱ期的CAGE基因启动子区的去甲基化直肠癌患者占24.6%(16/65),临床分期为Ⅲ-Ⅳ期的占75.4%(49/65);而甲基化的直肠癌患者中,临床分期为I-Ⅱ期的占66.7%(10/15),Ⅲ-Ⅳ期的占33.3%(5/15),两组间差异有统计学意义(P0.01)。结论:直肠癌组织和大肠腺瘤组织中,CAGE mRNA水平呈高表达。直肠癌的CAGE基因mRNA水平表达高于正常粘膜组织和大肠癌腺瘤组织。CAGE基因启动子区的去甲基化与淋巴结转移及临床分期密切相关。     

CDX2在胃癌及癌前病变组织中的表达及其临床意义

目的研究CDX2在胃癌及癌前病变组织中的表达及其与临床病理因素之间的关系。方法采用免疫组织化学S-P法检测CDX2在胃癌及癌前病变组织中的表达,采用阿新兰/过碘酸雪夫氏染色(AB/PAS)和高铁二铵/阿新兰(HID/AB)染色对胃黏膜肠化生进行分型。结果 (1)CDX2在正常胃黏膜组织、慢性浅表性胃炎中不表达,CDX2在肠化、异型增生及胃癌中的阳性率(81.7%、50%、48.2%)均明显高于正常胃黏膜组织(P0.05),在肠化组织中的表达明显高于异型增生和胃癌(P0.05),在异型增生和胃癌中的阳性率差异无显著性(P0.05)。CDX2的表达与肠化的分型和异型增生的分级之间均无关系。(2)CDX2在肠型胃癌中的阳性率(67.4%)明显高于弥漫型和混合型胃癌(25.7%、42.9%);CDX2的表达与胃癌分化程度呈正相关,而与淋巴结转移和临床分期呈负相关,差异均有统计学意义。CDX2阳性表达组的五年生存率明显高于阴性表达组(P=0.038);多种变量对胃癌患者术后生存时间影响的分析显示,CDX2的表达和淋巴结转移是影响胃癌预后的独立因素(P0.05)。结论 CDX2可能在胃粘膜肠化生的发生及胃癌的发生发展中起重要作用,可作为胃癌诊断、判断胃癌的恶性程度及患者预后的有用指标。     

NF2基因甲基化及其mRNA表达与乳腺癌发病的关系

目的:探讨乳腺癌中NF2基因启动子甲基化状态及其mRNA水平与乳腺癌发病的关系.方法:应用甲基化特异性聚合酶链反应(MSP)和逆转录-聚合酶链反应(RT-PCR)技术,检测47例乳腺癌组织及相应的癌旁组织和15例乳腺良性病变组织,分析NF2基因的甲基化与某些临床参数及mRNA表达的关系.结果:NF2基因启动子区在乳腺癌、癌旁和乳腺良性病变组织中的甲基化频率分别为57.4%(27/47)、23.4%(23/47)和0%(0/15).且乳腺癌组明显高于其余两组(P＜0.05).NF2基因发生甲基化与发病年龄、组织分型、转移和组织分级无相关性.乳腺癌组NF2基因mRNA的相对表达量(0.16±0.11)明显低于相应的癌旁组(0.27±0.14)及乳腺良性病变组(0.64±0.17)(P＜0.05).NF2基因启动子区甲基化频率与其mRNA表达呈负相关(Spearman's r=-0.314,P＜0.05).结论:NF2基因发生甲基化与乳腺癌的发生密切相关,NF2mRNA表达与NF2基因启动子高甲基化呈负相关.     

MGMT 甲基化影响替莫唑胺对胶质母细胞瘤疗效的研究

目的:探讨MGMT甲基化如何影响替莫唑胺对胶质母细胞瘤的治疗效果。方法:选取41个胶质母细胞瘤患者(根据相同替莫唑胺化疗方案治疗下临床结局的不同分为两组)的肿瘤组织,采用甲基化特异性聚合酶链反应分析胶质瘤组织中MGMT基因启动子区过甲基化状态,同时采用免疫组织化学法分析胶质瘤组织中MGMT蛋白表达情况。结果:临床结局不佳组中,以MGMT蛋白表达阳性的肿瘤为主(72.2%),而在结局相对良好组中,MGMT蛋白表达的阳性率仅为39.1%;在MGMT蛋白表达阳性的22例胶质母细胞瘤组织中,7例MGMT启动子甲基化,阳性率为31.8%,在MGMGT蛋白表达阴性的19例中,14例MGMT启动子甲基化,阳性率为73.7%(P<0.05)。结论:MGMT基因启动子区的甲基化状态与MGMT蛋白的表达相关。MGMT基因启动子过甲基化,MGMT蛋白表达较低;MGMT基因启动子去甲基化,MGMT蛋白表达较高。MGMT启动子过甲基化通过抑制MGMT基因的表达而增加替莫唑胺的疗效。     

胃癌组织中Bag-1蛋白表达特点及与临床病理学特征、预后的关系

目的:探讨Bc1-2相联系的抗凋亡基因1(Bag-1)蛋白在胃癌组织中的表达及与患者临床病理学特征、预后的关系。方法:选取病理科收集的79例胃癌组织及30例癌旁组织,标本收集时间2010年2月至2013年12月,采用免疫组化SP染色检测两组标本中的Bag-1蛋白表达水平,并分析Bag-1蛋白表达与胃癌患者临床病理学、预后的关系。结果:胃癌组织中的Bag-1蛋白阳性表达率65.82%显著的高于癌旁组织的13.33%(P0.05);Bag-1蛋白阳性表达的胃癌患者3年生存率30.77%显著的低于阴性表达患者的55.56%(P0.05);Bag-1蛋白阳性和阴性表达的患者1年以及2年生存率相差不大,差异无统计学意义(P0.05);Bag-1蛋白阳性表达的胃癌患者3年生存率30.77%显著的低于阴性表达患者的55.56%(P0.05)。结论:胃癌组织中的Bag-1蛋白高表达,并且与患者的胃癌临床分期和淋巴结转移以及预后有关。     

Fibulin3基因在非小细胞肺癌中的蛋白表达及甲基化检测

目的:检测Fibulin3基因在非小细胞肺癌患者(non-small cell hung cancer,NSCLC)组织中的表达和甲基化状态,并分析其临床病理意义.方法:收集59例NSCLC患者术后病理蜡块,进行免疫组化染色;提取癌组织及相应癌旁组织DNA,甲基化特异性聚合酶链反应(MSP)检测Fibulin3基因启动子区甲基化情况.结果:59例NSCLC标本中,25例(42.4%)Fibulin3表达水平比相应癌旁组织下调(P＜0.05);癌组织和相应癌旁组织甲基化22例和5例检出Fibulin3基因启动子区高甲基化,其阳性率分别为37.3%和8.5%,差异具有统计学意义(P＜0.001);Fibulin3启动子甲基化导致蛋白表达下调或缺失(P＜0.001),并与临床分期(P=0.035)及淋巴结转移(P=0.011)相关.结论:启动子甲基化引起的Fibulin3基因失活在NSCLC发生发展中起重要作用,Fibulin3启动子甲基化可能成为NSCLC早期诊断和预后评估的潜在标记物.     

E-cadherin和Cathepsin D的表达与乳腺导管癌的侵袭和淋巴结转移的关系

目的　观察上皮性钙粘素 (E cadherin)和组织蛋白酶D (CathepsinD)在乳腺导管癌中的表达并分析其与肿瘤侵袭及腋下淋巴结转移的关系。方法　应用免疫组织化学方法检测E cadherin和CathepsinD在乳腺导管癌组织中的表达。结果　乳腺导管原位癌 (carcinomainsitu ,CIS)组织中E cadherin的表达与浸润性导管癌 (infiltratingductalcarcinoma ,IDC)相比无明显差异 (P >0 0 5 )。乳腺导管癌腋下淋巴结阴性组 (nodenegativeductalcarcinoma,NNDC)中E cadherin的表达与腋下淋巴结阳性组 (nodepositiveductalcarcinoma ,NPDC)相比差异不明显 (P >0 0 5 )。乳腺导管癌间质中CathepsinD的表达CIS与IDC相比差异显著 (P <0 0 1) ,NNDC与NPDC相比差异显著 (P <0 0 5 ) ;而在癌细胞中CathepsinD的表达在上述两组中差异均不明显 (P >0 0 5 )。结论　E cadherin在乳腺导管癌的表达与肿瘤的侵袭及淋巴结转移无相关关系。CathepsinD在乳腺导管癌间质的表达与肿瘤的侵袭及淋巴结转移密切相关 ,可作为临床判定肿瘤恶性程度的一个参考指标。     

类风湿性关节炎患者外周血单个核细胞 IL-10 基因启动子甲基化状态研究

白细胞介素-10（interleukin-10,IL-10）是在类风湿性关节炎中发挥重要免疫调节作用的细胞因子,其基因失活与已分化的Th1和Th2细胞染色质结构重塑有关。为了探讨IL-10基因启动子甲基化及基因失活在类风湿性关节炎(Rheumatoid Arthritis,RA)发病和进展中的作用,采用逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)及甲基化特异性聚合酶链反应(MSP), 分别检测34例类风湿性关节炎患者和30例健康人外周血单个核细胞 IL-10 mRNA、蛋白表达水平及基因启动子甲基化状态。结果显示,病例组IL-10 mRNA及蛋白表达均低于健康对照组,无统计学差异（P>0.05）。病例组甲基化率(85.29%)高于健康对照组(43.33%), 具有统计学差异(x² =12.439,P=0.000)。IL-10基因启动子甲基化状态与其mRNA表达呈显著负相关(r=-0.579, P=0.001), 与所累关节数显著相关,但与血沉（ESR）、C反应蛋白（CRP）、类风湿因子(RF)、年龄均无相关性(P>0.05)。IL-10 mRNA表达与年龄、所累关节数、ESR、CRP及RF均无相关性(P>0.05)。上述结果提示,启动子甲基化可能是IL-10基因失活的重要机制,IL-10基因异常高甲基化状态可能参与了RA的发生发展。     

明胶酶及肿瘤抑制基因pten在喉鳞状细胞癌中表达的研究

目的探讨喉鳞癌中明胶酶MMP-2、MMP-9及肿瘤抑制基因PTEN的表达及其相关性.方法应用S-P免疫组化法检测68例喉鳞癌、9例异型增生及15例正常喉粘膜的MMP-2、MMP-9及PTEN蛋白表达.结果(1)MMP-2、MMP-9阳性表达率:喉鳞癌中分别为73.53%、79.41%,均显著高于异型增生表达率(44.44%、22.22%)及正常喉粘膜表达率(13.33%、20.00%),P<0.05;伴淋巴结转移的喉鳞癌组分别为96.88%、100%,显著高于未转移组52.78%、61.11%,P<0.01.T3-T4期MMP-9的表达(100%)高于T1-T2期(53.33%),P<0.01.(2)PTEN在异型增生喉上皮中高表达率66.67%,显著高于正常喉上皮(13.33%)P<0.01,喉鳞癌中高表达率44.12%,阴性表达率为22.06%,总异常率(66.17%)与正常组(13.33%)间差异显著,P<0.05.PTEN高表达率在伴淋巴结转移组(25.00%)低于未转移组(61.11%),P<0.05;与组织学分级负相关(P<0.01).(4)喉鳞癌中MMP-2与MMP-9表达间呈正相关,二者分别与PTEN表达呈负相关.结论MMP-2、MMP-9表达增强及PTEN表达减弱对喉鳞癌的浸润转移起促进作用且相互协同,是喉鳞癌侵袭的重要标志物,其中PTEN异常是一早期事件.     

胃癌中APC基因I1307K突变及蛋白质表达

为探讨肿瘤抑制基因APC结构及表达异常与胃癌发生、发展的关系,采用ARMS PCR检测胃癌中APC基因I1307K突变存在与否,免疫组织化学方法分析胃癌中APC蛋白表达水平。结果表明,在 62例胃癌高发区易感人群血液标本及45例胃癌中未检测到I1307K突变;胃癌（早期、进展期）中APC蛋白表达阳性率显著低于正常黏膜,进展期胃癌中APC蛋白表达阳性率显著低于早期胃癌,淋巴结转移阳性的胃癌中APC蛋白表达阳性率显著低于淋巴结转移阴性者。因此认为I1307K突变可能与国人胃癌发生无明显相关;APC蛋白低表达与胃癌发生、进展及淋巴结转移密切相关。 Abstract:In order to explore the correlation of the abnormalities of tumor suppressor gene APC with the carcinogenesis and progression of gastric cancer.The I1307K mutation of APC gene in gastric cancer was analysed using Amplification Refractory Mutation System PCR(ARMS ,PCR),also the expression of APC protein in gastric cancer of different stages was detected by immunohistochemical method.We found that there wasn't I1307K mutation of APC gene in 62 cases of blood samples of susceptible population in high incidence areas of gastric cancer and 45 cases of gastric cancer tissues.The positive rates of APC protein in gastric cancer (both early and progressive gastric cancer) were significantly lower than that in normal mucosa,the positive rates of APC protein in progressive gastric cancer were significantly lower than that in early gastric cancer,the positive rates of APC protein in gastric cancer with lymph node metastasis were significantly lower than that in gastric cancer without lymph node metastasis.So it was thought that there might be no correlation between the I1307K mutation of APC gene and carcinogenesis of gastric cancer in China,but the decreased expression of APC protein was closely related to the carcinogenesis,progression and lymph node metastasisof gastric cancer.     

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down‐regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase‐2, caspase‐9, and caspase‐3, cleavage of BID and inactivation of poly (ADP‐ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.     

ZIC1 is downregulated through promoter hypermethylation in gastric cancer

As one of major epigenetic changes responsible for tumor suppressor gene inactivation in the development of cancer, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes. In the current study we identified ZIC1 (Zic family member 1, odd-paired Drosophila homolog) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. In all of gastric cancer cells lines examined, ZIC1 expression was downregulated and such downregulation was accompanied with the hypermethylation of ZIC1 promoter. Demethylation treatment with 5-aza-2′-deoxycytidine (Aza) reversed ZIC1 downregulation, highlighting the importance of promoter methylation to ZIC1 downregulation in gastric cancer cells. Notably, ZIC1 expression was significantly downregulated in primary gastric carcinoma tissues in comparison with non-tumor adjacent gastric tissues (p < 0.01). Accordingly, promoter methylation of ZIC1 was frequently detected in primary gastric carcinoma tissues (94.6%, 35/37) but not normal gastric tissues, indicating that promoter hypermethylation mediated ZIC1 downregulation may play an important role in gastric carcinogenesis. Indeed, ectopic expression of ZIC1 led to the growth inhibition of gastric cancer cells through the induction of S-phase cell cycle arrest (p < 0.01). Our results revealed ZIC1 as a novel candidate tumor suppressor gene downregulated through promoter hypermethylation in gastric cancer.     

p16 hypermethylation during gastric carcinogenesis of Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine

Inactivation of the tumor suppressor gene, p16 by CpG hypermethylation is a common event in various tumors including gastric carcinoma. The aim of this study is to investigate if p16 hypermethylation is an early and frequent event in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The frequency and timing of p16 hypermethylation during the multistep gastric carcinogenesis in Wistar rats were analyzed in various microdissected gastric lesions. The p16 methylation status and the presence of p16 protein were analyzed by methylation-specific PCR and immunohistochemistry, respectively. Results showed that p16 methylation frequency was correlated with the severity of gastric pathologic lesions, positively. For instance, p16 methylation was found in 2.7% of normal gastric epithelium (n = 36), 16.7% of chronic atrophy gastritis (n = 24), 37.5% of dysplasia (n = 24), 67.4% of gastric adenoma (n = 43), and 85.2% of gastric carcinoma (n = 27). The p16 methylation in the distal stomach epithelium was higher than that in the proximal stomach. p16 protein was expressed in all of 15 p16 unmethylated gastric epithelial samples, but not expressed in all of 12 p16 methylated samples. These results suggest that CpG island hypermethylation may account for the silencing of p16 in rat stomach and is an early event whose accumulation will finally lead to gastric carcinogenesis.     

Aberrant hypermethylation of the FGFR2 gene in human gastric cancer cell lines

We have previously shown that fibroblast growth factor receptor 2 (FGFR2) plays an important role in gastric carcinogenesis. In this study, we assessed DNA methylation status in the promoter region of FGFR2 gene in gastric cancer cell lines, and indicated that this region was highly methylated, compared with FGFR2-expressing gastric cancer cell lines. Moreover, the restoration of FGFR2 expression by treating methylated cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine strongly suggests that the loss of FGFR2 expression may be due to the aberrant hypermethylation in the promoter region of the FGFR2 gene. Thus, our results suggest that the epigenetic silencing of FGFR2 through DNA methylation in gastric cancer may contribute to tumor progression.     

Delivery of BR2‐SOX17 fusion protein can inhibit cell survival,proliferation, and invasion in gastric cancer cells through regulating Klotho gene expression

The prognosis of advanced gastric cancer is poor and understanding the biology and subsequent development of new targeting therapy is still an urgent need. This study was conducted to explore the effect of BR2 (a 17‐amino acid peptide)‐SOX17 (human sex determining region Y (SRY)‐related high‐mobility group (HMG) box protein family member 17) fusion protein on Klotho gene expression in gastric cancer cells. The regulatory effects of SOX17 on Klotho gene in gastric cancer cells were tested using dual‐luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. The therapeutic effects of BR2‐SOX17 were evaluated by proliferation, colony formation, invasion assay, and cell apoptosis analysis. Results showed that SOX17 enhanced Klotho gene expression in gastric adenocarcinoma cells through binding to the promoter of Klotho gene. BR2‐SOX17 fusion protein was effective in delivering SOX17 into gastric cancer cells and subsequently inhibited the cell proliferation, colony formation, and invasion, increased E‐cadherin protein expression, decreased vimentin protein expression, as well as induced apoptosis. Our findings suggested SOX17 can bind to the promoter of Klotho gene to enhance Klotho gene expression in gastric cancer cells. The fused BR2‐SOX17 protein is an effective agent for targeting therapy of gastric cancer.     

Mitochondrial microsatellite instability in gastric cancer and gastric epithelial dysplasia as a precancerous lesion

Background: Genetic instability in gastric cancer represents a key molecular step that occurs early in the carcinogenesis process. To clarify the role of genetic instability in the progression from gastric dysplasia to gastric cancer, mitochondrial microsatellite instability (mtMSI) was studied in gastric cancer and gastric dysplasia. Methods: DNA was isolated from paired normal and tumoral tissues in 24 patients with gastric dysplasia (low grade) and 49 patients with gastric cancer. mtMSI was analyzed using eight microsatellite markers. mtMSI in gastric dysplasia was studied prospectively to elucidate the relation between mtMSI and gastric carcinogenesis. Results: mtMSI was found in 5 (10.2%) of 49 gastric cancer patients. The mtMSI phenotype was not associated with age, gender, and Helicobacter pylori infection. However, all of the mtMSI was found in intestinal-type gastric cancer (20.8%, p = 0.02). In gastric dysplasia, mtMSI was detected in 3 (12.5%) of 24 patients with gastric dysplasia. mtMSI-positive gastric dysplasia showed a poor prognosis statistically compared to mtMSI negative through progression to high-grade dysplasia or gastric cancer. Conclusions: These data suggest that mtMSI may be an early and important event in the progression of gastric carcinogenesis, especially in intestinal-type gastric cancer.