Prognostic significance of aberrant gene methylation in gastric cancer

Promoter methylation acts as an important alternative to genetic alterations for gene inactivation in gastric carcinogenesis. Although a number of gastric cancer-associated genes have been found to be methylated in gastric cancer, valuable methylation markers for early diagnosis and prognostic evaluation of this cancer remain largely unknown. In the present study, we used methylation-specific PCR (MSP) to analyze promoter methylation of 9 gastric cancer-associated genes, including MLF1, MGMT, p16, RASSF2, hMLH1, HAND1, HRASLS, TM, and FLNc, and their association with clinicopathological characteristics and clinical outcome in a large cohort of gastric cancers. Our data showed that all of these genes were aberrantly methylated in gastric cancer, ranging from 8% to 51%. Moreover, gene methylation was strongly associated with certain clinicopathological characteristics, such as tumor differentiation, lymph node metastasis, and cancer-related death. Of interest, methylation of MGMT, p16, RASSF2, hMLH1, HAND1, and FLNc was closely associated with poor survival in gastric cancer, particularly MGMT, p16, RASSF2 and FLNc. Thus, our findings suggested these epigenetic events may contribute to the initiation and progression of gastric cancer. Importantly, methylation of some genes were closely relevant to poor prognosis in gastric cancer, providing the strong evidences that these hypermethylated genes may be served as valuable biomarkers for prognostic evaluation in this cancer.     

Silencing NKD2 by promoter region hypermethylation promotes gastric cancer invasion and metastasis by up-regulating SOX18 in human gastric cancer

Naked cuticle homolog2 (NKD2) is located in chromosome 5p15.3, which is frequently loss of heterozygosity in human colorectal and gastric cancers. In order to understand the mechanism of NKD2 in gastric cancer development, 6 gastric cancer cell lines and 196 cases of human primary gastric cancer samples were involved. Methylation specific PCR (MSP), gene expression array, flow cytometry, transwell assay and xenograft mice model were employed in this study. The expression of NKD1 and NKD2 was silenced by promoter region hypermethylation. NKD1 and NKD2 were methylated in 11.7% (23/196) and 53.1% (104/196) in human primary gastric cancer samples. NKD2 methylation is associated with cell differentiation, TNM stage and distant metastasis significantly (all P < 0.05), and the overall survival time is longer in NKD2 unmethylated group compared to NKD2 methylated group (P < 0.05). Restoration of NKD2 expression suppressed cell proliferation, colony formation, cell invasion and migration, induced G2/M phase arrest, and sensitized cancer cells to docetaxel. NKD2 inhibits SOX18 and MMP-2,7,9 expression and suppresses BGC823 cell xenograft growth. In conclusion, NKD2 methylation may serve as a poor prognostic and chemo-sensitive marker in human gastric cancer. NKD2 impedes gastric cancer metastasis by inhibiting SOX18.     

Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer

The role of secreted frizzled-related protein (SFRP) genes in gastric cancer remains largely unknown. We determined the frequency and functional significance of SFRPs hypermethylation in human gastric cancer. The expression and methylation status of four SFRP members (SFRP1, 2, 4, and 5) in primary gastric cancer samples was screened. The biological effects of SFRP were analysed by flow cytometry, cell viability assay and in vivo tumour growth in nude mice. Among the four SFRPs, only SFRP2 was significantly downregulated in gastric cancer as compared to adjacent non-cancer samples (P<0.01). Promoter hypermethylation of SFRP2 was detected in 73.3% primary gastric cancer tissues, 37.5% of samples showing intestinal metaplasia and 20% adjacent normal gastric tissues. Bisulphite DNA sequencing confirmed the densely methylated SFRP2 promoter region. Demethylation treatment restored the expression of SFRP2 in gastric cancer cell lines. Forced expression of SFRP2 induced cell apoptosis, inhibited proliferation of gastric cancer cells and suppressed tumour growth in vivo. Moreover, methylated SFRP2 was detected in 66.7% of serum samples from cancer patients but not in normal controls. In conclusion, epigenetic inactivation of SFRP2 is a common and early event contributing to gastric carcinogenesis and may be a potential biomarker for gastric cancer.     

Epigenetic inactivation and tumor suppressor activity of HAI‐2/SPINT2 in gastric cancer

Hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI‐2/SPINT2) encodes Kunitz‐type protease inhibitor that regulates HGF activity. Inspection of the human HAI‐2/SPINT2 locus uncovered a large and dense CpG island within the 5′ region of this gene. Analysis of cultured human gastric tumor lines indicated that HAI‐2/SPINT2 expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5‐aza‐2′‐deoxycytidine. Bisulfite DNA sequencing confirmed the densely methylated HAI‐2/SPINT2 promoter region. Forced expression of HAI‐2/SPINT2 induced cell apoptosis, suppressed anchorage independent growth in vitro and tumor growth in vivo. We investigated HAI‐2/SPINT2 aberrant methylation in patients with gastric cancer. The HAI‐2/SPINT2 methylation was found preferentially in cancerous tissues (30 of 40, 75%) compared with nontumor tissues (no methylation was detected), indicating that this aberrant characteristic is common in gastric malignancies. In conclusion, epigenetic inactivation of HAI‐2/SPINT2 is a common event contributing to gastric carcinogenesis and may be a potential biomarker for gastric cancer.     

RASAL2 对胃癌细胞侵袭与上皮间质转化的影响*

目的：探讨RASAL2 表达改变对胃癌细胞侵袭与迁移能力的影响,以及对细胞上皮间质转化（epithelial-mesenchymal transition ,EMT ）的影响。方法：以胃癌细胞系SNU-1、SNU-16与AGS 为体外模型,通过RNA 干扰技术构建RASAL2 敲低细胞系,通过过表达慢病毒载体构建RASAL2 过表达细胞系。利用体外侵袭与迁移实验,划痕实验观察RASAL2 表达改变对两种胃癌细胞系侵袭与迁移能力的影响。同时利用免疫显微荧光技术与免疫印迹分析RASAL2 表达改变对胃癌细胞EMT 的影响。利用免疫印记分析RASAL2 对Ras-ERK通路的活化影响。结果：两种不同靶位的RASAL2-shRNA 均可以有效抑制RASAL2 在SNU-16与AGS 细胞中的表达。细胞体外侵袭与迁移能力在RASAL2 敲低后显著高于对照组。划痕实验也提示同样结果。上皮标志 E-cadherin表达在RASAL2 敲低后发生下调,同时间质标志Vimentin 发生上调。EMT 调控转录因子Snail 也发生上调。Ras-ERK 通路活化在RASAL2 敲低后显著上调。RASAL2 过表达抑制SNU-1 细胞迁移能力。结论：RASAL2 在胃癌中具有抑制细胞侵袭与迁移的能力。同时也是胃癌细胞EMT 的调控分子。RASAL2 发挥生物学功能至少是部分依赖Ras-ERK 通路的。     

CDKL1对胃癌细胞迁移及侵袭的作用

目的:探讨细胞周期素依赖激酶样蛋白1(CDKL1)对胃癌细胞系SGC7901迁移及侵袭的作用.方法:将胃癌细胞系SGC7901分为实验组及对照组,采用慢病毒转染siRNA至胃癌细胞构成实验组,转染无意义序列作为对照组.通过细胞划痕实验检测胃癌SGC7901细胞的迁移能力;Transwell小室实验检测其侵袭能力;Western blot检测CDKL1低表达后对胃癌细胞中AEG-1和MMP-9蛋白表达的影响.结果:与对照组相比,CDKL1低表达后实验组细胞迁移及侵袭能力明显降低(P＜0.05),实验组胃癌细胞中AEG-1和MMP-9蛋白的表达水平明显下降(P＜0.05).结论:特异性下调CDKL1基因可抑制胃癌细胞的迁移及侵袭能力,表明CDKL1基因可促进胃癌细胞的侵袭以及转移,并且与AEG-1和MMP-9密切相关.     

Upregulation of LINC00659 expression predicts a poor prognosis and promotes migration and invasion of gastric cancer cells

Long non-coding RNAs (lncRNAs) serve an important role in the progression of cancer. LINC00659 was recently identified as a novel oncogenic lncRNA involved in colon cancer cell proliferation via modulating the cell cycle. However, the function of LINC00659 in other types of cancer, especially in gastric cancer (GC), remains unknown. In the present study, bioinformatics analysis combined with cell experiments were performed to explore the function of LINC00659 in GC. It was revealed that LINC00659 expression was significantly upregulated in GC tissues and cell lines. Increased levels of LINC00659 were associated with advanced tumor stage and unfavorable prognosis of patients with GC. Additionally, upregulated LINC00659 expression promoted the migration and invasion of GC cells. Further analysis using a bioinformatics method revealed that matrix metalloproteinase 15 and IQ motif-containing GTPase activating protein 3 were potential downstream targets of LINC00659 involved in tumor metastasis, although the precise underlying mechanism requires further exploration.     

CPNE7通过上皮间质转化影响胃癌细胞增殖、迁移和侵袭

目的:探讨CPNE7在胃癌组织和细胞中的表达及影响胃癌细胞增殖、迁移和侵袭的机制。方法:基于生物信息学数据库GEPIA 从转录水平分析CPNE7在胃癌及癌旁正常组织中的表达差异。qRT-PCR法检测CPNE7在3种胃癌细胞系(AGS、MKN-45、HGC-27)和胃黏膜正常上皮细胞(GES-1)中的表达,选择表达量相对较高的AGS细胞作为后续实验细胞。通过慢病毒转染AGS细胞敲减CPNE7的表达,根据不同处理分为RNAi-vector组、RNAi-1组、RNAi-2组、RNAi-3组,qRT-PCR、Western blot验证细胞转染效率,选择敲减效率最有效的RNAi-1(实验组)和RNAi-vector(对照组)进行后续实验。采用CCK-8法检测细胞活力,EdU和克隆形成实验检测细胞增殖和克隆形成能力,划痕愈合实验和Transwell实验检测细胞迁移与侵袭能力,Western blot检测细胞凋亡相关蛋白(Caspase3、Caspase7、Caspase9、Bax、Bcl-2)及上皮间质转化（epithelial-mesenchymal transition,EMT)相关蛋白（E-cadherin、N-cadherin、Vimentin)的表达水平。结果:CPNE7在胃癌组织中的表达明显高于正常组织(P＜0.05)。与正常胃黏膜上皮细胞(GES-1)相比较,CPNE7在胃癌细胞中明显高表达(P＜0.05),在AGS胃癌细胞中CPNE7相对表达量最高（P＜0.001)。与RNAi-vector组相比较,敲减CPNE7可抑制胃癌细胞的增殖、迁移和侵袭（P＜0.05)。与RNAi-vector组相比较,敲减CPNE7可上调细胞凋亡相关蛋白Caspase3、Caspase7、Caspase9、Bax的表达,下调Bcl-2的表达（P＜0.05)。与RNAi-vector组相比较,敲减CPNE7可增加上皮标志物蛋白(E-cadherin)的表达水平(P＜0.01),降低间质标志物蛋白(N-cadherin、Vimentin)的表达水平（P＜0.01)。结论:CPNE7在胃癌组织和细胞中表达上调,敲减CPNE7可通过影响EMT进程抑制胃癌细胞增殖、迁移与侵袭,其可能成为胃癌的潜在分子标志物。     

胰岛素样生长因子Ⅱ mRNA结合蛋白3在肺腺癌中的表达及临床意义

目的:研究胰岛素样生长因子ⅡmRNA结合蛋白3(IMP3)在肺腺癌患者中的表达及其临床意义。方法:采用免疫组织化学方法检测92例肺腺癌患者的标本及30例癌旁正常标本中IMP3的表达,并分析与患者的年龄、性别、病理分级、TNM分期、远处转移、肿瘤的大小及与预后的关系。结果:IMP3在肺腺癌组中的阳性表达率为56.5%,明显高于对照组(均为阴性),差异具有统计学意义(P<0.001);IMP3在肺腺癌中的表达与患者的年龄、性别以及肿瘤的大小无关(P>0.05);与病理分级(P<0.001)、临床分期(P<0.05)及是否有远处转移有关(P<0.05);IMP3表达与肺腺癌患者术后3年生存率呈负相关(P<0.001)。结论:IMP3在肺腺癌组织中表达,且与肺腺癌的病理分级、临床分期及远处转移有关。     

MicroRNA-212 functions as an epigenetic-silenced tumor suppressor involving in tumor metastasis and invasion of gastric cancer through down-regulating PXN expression

Altered expression of paxillin (PXN) is closely linked to the pathogenesis progression, metastasis and prognosis of different malignancies including gastric cancer (GC). Epigenetic silencing of tumor-suppressive microRNAs (miRNAs) is a crucial component of the mechanism underlying activation of oncogenes in tumor. To screen for epigenetically silenced miRNAs which target PXN in GC, we performed bioinformatics algorithms and real-time PCR analysis, and identified miR-212 as the optimum candidate gene. A luciferase reporter gene assay validated that miR-212 directly targets the 3’UTR region of PXN. Importantly, miR-212 levels were inversely correlated with PXN expression in GC cell lines and clinical tumor tissues. The use of miR-212 minics decrease PXN mRNA and protein level in GC cell lines. Moreover, low expression of miR-212 and its promoter hypermethylation were causally related and were associated with aggressive tumor phenotype and adverse prognosis in GC. Restoring mir-212 expression by exogenous mirprecursor molecules transfection or reexpression of endogenous miR-212 treated by 5-aza-2’-deoxycytidine (5-aza) can exert similar effect that reduce GC cells invasion and metastasis abilities in vitro by interacting PXN gene. In addition, 5-aza-induced PXN reduction could be partically blocked by miR-212 inhibitor, resulting in a reversal of weankening cell migration and invasion ability of 5-aza. A rescue experiment and a loss-of-function experiment in vitro and vivo showed that PXN restoration rescues migration and invasion phenotype in miR-212 overexpressed GC cell lines and PXN knockdown blocks GC cells migration and invasion in the presence miR-212 inhibitors. Taken together, our results clearly show that overexpression of PXN induced by methylationsuppressed miR-212 promotes tumor metastasis and invasion, and regulation of miR-212 expression may be a novel therapeutic strategy for gastric cancer.     

胃癌中PCDH8基因启动子甲基化状态及其临床意义

目的:本研究旨在明确胃癌中PCDH8表达水平以及PCDH8基因启动子甲基化情况,分析PCDH8的甲基化差异表达与胃癌临床病理因素的关系。方法:我们通过RT-PCR、Western blot方法检测了65例胃癌组织标本和30例正常胃黏膜标本中PCDH8 mRNA及蛋白的表达,然后分析了其在不同组织中的表达差异。并运用甲基化特异性PCR(MSP)技术检测PCDH8基因启动子区在不同组织中的甲基化情况,并分析其基因启动子区甲基化情况与胃癌临床病理因素的关系。结果:PCDH8 mRNA、蛋白在正常胃组织中为阳性表达,而在胃癌组织中则表达缺失或下调,其差异有统计学意义(P<0.05)。PCDH8甲基化情况在胃癌组织(55.38％,36/65)与正常组织(0％,0/65)、癌旁组织(41.54％,27/65)与正常组织间存在显著差异（P< 0.05)。65例胃癌患者中,其甲基化情况与胃癌患者性别、年龄、肿瘤大小、远处转移及TNM分期均无关(P>0.05)。多因素回归分析结果表明,淋巴结转移为PCDH8基因启动子区甲基化的独立因素(P=0.026,95%CI 1.12~86.84)。结论:PCDH8甲基化及表达情况与胃癌患者肿瘤分化程度及淋巴结转移有关。PCDH8在调节胃癌的发生和转移过程中具有重要作用,它有可能作为胃癌的抑癌基因而发挥作用,并作为标记物对胃癌的诊断、预后判定及治疗提供研究方向。     

TEM7 (PLXDC1), a key prognostic predictor for resectable gastric cancer,promotes cancer cell migration and invasion

Tumor endothelial marker 7 (TEM7) is a new candidate of molecular target for antiangiogenic therapy. This study aims to evaluate its expression in gastric cancer (GC) and to explore the correlation between its expression and the clinical outcome of patients. Expression of TEM7 was analyzed in both tumor tissues and cell lines of GC by real-time quantitative RT-PCR (qRT-PCR) and Western blot. RNA interference (RNAi) approaches were used to investigate the biological functions of TEM7. The effects of TEM7 on cell migration and invasion were evaluated by Transwell assays. In vitro experiments revealed that TEM7 was significantly overexpressed in GC cell lines (N87, AGS and SGC-7901) by 2-fold to 4-fold, and knockdown of TEM7 could significantly inhibit cancer cell migration and invasion. For GC patients, TEM7 gene expression was elevated in tumors in most cases (25/31), and its expression was closely correlated with tumor differentiation, depth of cancer invasion, lymphatic metastasis and TNM stage. The overall survival of TEM7 (-) group was significantly higher than that of TEM7 (+) group (P = 0.048) and TEM7 (++) group (P = 0.003). TEM7 is highly expressed in GC and is likely correlated with tumor invasion and migration, and thus its expression is closely related to the clinical outcome of patients.     

RhoGDI2 promotes epithelial-mesenchymal transition via induction of Snail in gastric cancer cells

Rho GDP dissociation inhibitor 2 (RhoGDI2) expression correlates with tumor growth, metastasis, and chemoresistance in gastric cancer. Here, we show that RhoGDI2 functions in the epithelial-mesenchymal transition (EMT), which is responsible for invasiveness during tumor progression. This tumorigenic activity is associated with repression of E-cadherin by RhoGDI2 via upregulation of Snail. Overexpression of RhoGDI2 induced phenotypic changes consistent with EMT in gastric cancer cells, including abnormal epithelial cell morphology, fibroblast-like properties, and reduced intercellular adhesion. RhoGDI2 overexpression also resulted in decreased expression of the epithelial markers E-cadherin and β-catenin and increased expression of the mesenchymal markers vimentin and fibronectin. Importantly, RhoGDI2 overexpression also stimulated the expression of Snail, a repressor of E-cadherin and inducer of EMT, but not other family members such as Slug or Twist. RNA interference-mediated knockdown of Snail expression suppressed RhoGDI2-induced EMT and invasion, confirming that the effect was Snail-specific. These results indicate that RhoGDI2 plays a critical role in tumor progression in gastric cancer through induction of EMT. Targeting RhoGDI2 may thus be a useful strategy to inhibit gastric cancer cell invasion and metastasis.     

Clinicopathologic study of advanced gastric cancer without serosal invasion in young and old patients

Fifty-seven patients treated by radical gastric resections were retrospectively studied to understand the clinicopathologic characteristics of advanced gastric cancer without serosal invasion (the depth of tumor invasion limited to the muscularis propria or subserosal layer) in young and old age persons. There were 36 patients in the old age group (age > 60 years) and 21 in the young age group (age ≤ 40 years). The clinical and pathologic parameters for this study included sex, gross type, location, maximum tumor size, depth of invasion, lymph node metastasis, tumor stage, histologic type, and rate of curative resection. The old patients had a higher percentage of small tumors, subserosal invasion and lymph node metastasis, but these parameters were not significantly different from those of the young patients, nor did the sex ratio, gross type, location, and rate of curative resection show significant differences. The histologic feature was the only statistically significant parameter, determined by univariate and multivariate analyses. Poorly differentiated adenocarcinoma and signet ring cell carcinoma were detected in 10 (47.6%) and 4 (19.0%) of the 21 younger patients, respectively, while there were 4 (11.1%) and 2 (5.6%) in the old age group. Although the gastric cancer in young patients had more aggressive histologic characteristics than it did in elderly patients, survival rates between the two groups did not differ to any great degree. Our findings indicate that the prognosis for younger patients with advanced gastric cancer without serosal invasion was favorable when curative resection was performed. © 1996 Wiley-Liss, Inc.     

KRAS mutations and CDKN2A promoter methylation show an interactive adverse effect on survival and predict recurrence of rectal cancer

Colonic and rectal cancers differ in their clinicopathologic features and treatment strategies. Molecular markers such as gene methylation, microsatellite instability and KRAS mutations, are becoming increasingly important in guiding treatment decisions in colorectal cancer. However, their association with clinicopathologic variables and utility in the management of rectal cancer is still poorly understood. We analyzed CDKN2A gene methylation, CpG island methylator phenotype (CIMP), microsatellite instability and KRAS/BRAF mutations in a cohort of 381 rectal cancers with extensive clinical follow‐up data. BRAF mutations (2%), CIMP‐high (4%) and microsatellite instability‐high (2%) were rare, whereas KRAS mutations (39%), CDKN2A methylation (20%) and CIMP‐low (25%) were more common. Only CDKN2A methylation and KRAS mutations showed an association with poor overall survival but these did not remain significant when analyzed with other clinicopathologic factors. In contrast, this prognostic effect was strengthened by the joint presence of CDKN2A methylation and KRAS mutations, which independently predicted recurrence of cancer and was associated with poor overall and cancer‐specific survival. This study has identified a subgroup of more aggressive rectal cancers that may arise through the KRAS‐p16 pathway. It has been previously shown that an interaction of p16 deficiency and oncogenic KRAS promotes carcinogenesis in the mouse and is characterized by loss of oncogene‐induced senescence. These findings may provide avenues for the discovery of new treatments in rectal cancer.     

Flotillin-2在胃癌组织中的表达及其临床意义

[摘要] 目的：检测脂筏标记蛋白-2(Flotillin-2,Flot-2)在胃癌组织中的表达水平,分析Flot-2 的表达与胃癌临床病理特征及预后的关系。方法：选取南昌大学第二附属医院胃肠外科于2009 年1 月至2010 年4 月行手术切除的胃癌组织112 例及与其配对的癌旁组织,采用免疫组织化学法检测肿瘤组织中Flot-2 的表达水平,采用Spearman 检验Flot-2 表达与胃癌临床病理特征及预后的关系,采用Kaplan-Meier 法及Log-Rank 检验分析其生存数据。结果：Flot-2 阳性表达呈黄色颗粒,主要在细胞质中表达,胃癌组织中的表达量明显高于癌旁组织（53.57% vs 46.43%,P<0.05）。Flot-2 表达与患者的性别、年龄、肿瘤位置、分化程度无显著关联（P>0.05）,与肿瘤大小、浸润深度、淋巴结转移、远处转移及AJCC分期均显著关联（均P<0.01）。Flot-2 低表达组患者的5 年总体生存率明显高于高表达组（P<0.01）。Cox 回归分析显示,远处转移、AJCC分期和Flot-2 蛋白表达水平为胃癌患者预后的独立危险因素。结论：胃癌组织中高表达Flot-2,其与患者不良预后密切相关,是胃癌预后的独立危险因素,有望成为胃癌治疗的潜在新靶点。