多基因甲基化检测对结直肠癌早期诊断的意义

目的 通过对结直肠癌（CRC）组织中多基因甲基化水平的检测,探讨多基因甲基化对CRC早期诊断的意义.方法 选取25例CRC患者胃镜采集标本组织,通过甲基化特异性PCR（MSP）检测CRC和炎性组织中甲基化水平,分析其与CRC患者临床各指标的关系.结果 在CRC患者中,APC、P16、MLH1、DCC甲基化率分别为52.0%（13/25）、32.0%（8/25）、44.0%（11/25）、60.0%（15/25）.炎性组织中仅有1例DCC甲基化异常表达,甲基化率为10.0%（1/10）,甲基化阳性率显著高于炎性组织（P＜0.05）,4种基因甲基化联合检测诊断CRC的阳性率为92.0%（23/25）,高于单个基因检测的甲基化阳性率,差异有统计学意义（P＜0.01）.结论 APC、P16、MLH1、DCC基因在CRC患者中甲基化水平异常表达,联合检测APC、P16、MLH1、DCC可能作为CRC早期诊断的标志物.     

Downregulation of fibulin-3 gene by promoter methylation in colorectal cancer predicts adverse prognosis

Fibulin-3 gene has been identified as an antagonist of angiogenesis. We investigated the protein expression and promoter methylation status of fibulin-3 gene in colorectal cancer (CRC) and analyzed its correlation with clinicopathological factors. The study population enrolled 85 paired CRC specimens and adjacent normal tissues, as well as 32 cases of colorectal adenoma. Genomic DNA was extracted from paraffin-embedded samples using manual microdissection. Methylation-specific polymerase chain reaction (MSP) was used to determine the promoter methylation status and fibulin-3 gene expression was detected by immunohistochemistry. The results showed that, downregulation or silence of fibulin-3 protein was found in 57.6% (49/85) of CRC tissues, which was significantly higher than that of adjacent normal tissues (28.2%, 24/85) and colorectal adenoma (34.4%, 11/32) (P<0.05). Furthermore, 33 out of 85 (38.8%) CRC specimens showed hypermethylation in fibulin-3 promoter region, and fibulin-3 methylation was closely correlated with its loss of expression. Also, downregulation of fibulin-3 was associated with advanced stage (P=0.008) and lymph node metastasis (P=0.013). Survival analyses and Cox proportional hazard models indicated that fibulin-3 downregulation was an independent factor related to adverse overall survival (OS) and disease-free survival (DFS) of CRC. In conclusion, we found aberrant methylation caused fibulin-3 downregulation in CRC, and fibulin-3 downregulation was correlated with tumor stage, lymph node metastasis and poor survival, which maybe use as a potential prognostic factor for CRC.     

IL-6表观遗传改变调节SOCS3在大肠癌发病机制中的作用

目的 检测大肠癌（CRC）中IL-6、SOCS3基因mRNA表达和启动子甲基化状态,以及STAT3基因mRNA表达水平,探讨大肠癌中IL-6、SOCS3基因表观遗传学改变及其对IL-6/STAT3通路的影响.方法 实时荧光定量PCR检测20例CRC癌组织及癌旁组织IL-6、SOCS3及STAT3基因mRNA水平,甲基化特异性PCR（MSP）检测SOCS3甲基化状态,亚硫酸氢盐测序（BSP）检测IL-6启动子甲基化状态.结果 与癌旁组织相比,癌组织中IL-6、STAT3 mR-NA表达显著增高（P＜0.05）,而SOCS3显著降低,IL-6与SOCS3表达呈负相关;癌组织中IL-6启动子-633、-611、-575、-575bp位点甲基化水平均降低,而SOCS3则呈现高度甲基化状态（P＜0.05）.结论 大肠癌中IL-6和SOCS3基因DNA甲基化状态异常,影响基因表达,从而诱导IL-6/STAT3信号转导通路持续活化,促进了大肠癌的发生发展.     

Expression and clinical significance of SYNE1 and MAGI2 gene promoter methylation in gastric cancer

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related mortality globally. Abnormal DNA methylation is closely related to gastric cancer. The purpose of the study was to investigate the methylation of the SYNE1 and MAGI2 gene promoter and its relationship with the clinical-pathological factors, chemotherapy efficacy, and survival, thus providing a new biomarker for the prognosis and chemotherapy efficacy in gastric cancer.The methylation status of SYNE1 and MAGI2 in gastric cancer and adjacent tissues was detected by MSP method in 70 cases of advanced gastric cancer paraffin specimens.The methylation rate of the SYNE1 and MAGI2 gene promoter region was higher in gastric cancer tissues compared with adjacent tissues. The methylation status of SYNE1 was associated with the age at diagnosis and the size of the primary tumors, but no clinical or pathological factors have been found to be related with the methylation status of MAGI2 promoter. A high level of SYNE1 promoter methylation was associated with poorer chemotherapy efficacy in recurrent patients with gastric cancer. Thirty-three percent of the 70 patients exhibited highly methylated MAGI2; in this group, the median progression-free survival time was 4.1 months, shorter than those with negative methylated MAGI2 whose PFS was 5.1 months.MAGI2 is more methylated in gastric cancer than in adjacent tissues suggesting that hypermethylation changes in MAGI2 may be one of the mechanisms of tumorigenesis in gastric cancer. The methylation status of the SYNE1 and MAGI2 promoter regions may affect the chemotherapy efficacy of advanced gastric cancer. The prognosis of MAGI2-negative patients was better than that of positive ones, suggesting that MAGI2 may be an independent prognostic factor for PFS in patients with advanced gastric cancer.     

Epigenetic silencing of LRRC3B in colorectal cancer

Objective. Tumor suppressor gene silencing via promoter hypermethylation is an important event in the pathogenesis of colorectal cancer (CRC). Some aberrant DNA hypermethylation has high tumor specificity, so it may contribute to early diagnosis of CRC. The objective of this study was to establish novel therapeutic and diagnostic strategies against CRC by identifying the novel methylation-related genes. Material andmethods. Two microarray-based approaches were used to identify novel methylation-related genes in CRC. We identified methylation-sensitive genes in colon cancer cell line SW1116 by comparing differential expression genes after treatment with the methylation inhibiting drug, 5-aza-2'-deoxycitidine (5-aza-dC) using gene expression microarray. Promoter microarray analysis was performed to identify cancer-specific, methylation-related genes in two patients with CRC. Gene promoter methylation was identified by methylation-specific polymerase chain reaction (PCR) (MSP) in primary CRC. Gene expression level was assessed using real-time PCR analysis. Results. By using gene expression microarray, up-regulation of 253 genes was detected in the CRC cell line, SW1116, after treatment with 5-aza-dC. Of the 253 genes identified by gene expression microarray analysis, LRRC3B (leucine-rich repeat containing 3B) was isolated as a potential methylation-specific gene by promoter microarray analysis. MSP analysis showed frequent methylation of LRRC3B in primary CRC (24/31 cases, 77%). In addition, the LRRC3B methylation intensity was significantly higher in cancer tissues than in the corresponding non-cancerous tissues. Decreased LRRC3B expression (17/31, 55%) was observed in the cancer tissues by real-time PCR. Conclusions.LRRC3B may be a novel methylation-sensitive tumor suppressor gene in CRC. LRRC3B methylation has significant tumor specificity and may be a biomarker of CRC.     

Hypermethylated SFRP2 gene in fecal DNA is a high potential biomarker for colorectal cancer noninvasive screening

AIM： To investigate the feasibility of detecting hypermethylated secreted frizzled-related protein 2 （SFRP2） gene in fecal DNA as a non-invasive screening tool for colorectal cancer （CRC）. METHODS： Fluorescence-based real-time PCR assay （MethyLight） was performed to analyze SFRP2 gene promoter methylation status in a blinded fashion in tumor tissues and in stool samples taken from 69 CRC patients preoperatively and at the 9th postoperative day, 34 patients with adenoma ≥1 cm, 26 with hyperplastic polyp, and 30 endoscopically normal subjects. Simultaneously the relationship between hypermethylation of SFRP2 gene and clinicopathological features was analyzed. RESULTS： SFRP2 gene was hypermethylated in 91.3% （63/69） CRC, 79.4% （27/34） and 53.8% （14/26） adenoma and hyperplastic polyp tissues, and in 87.0% （60/69）, 61.8% （21/34） and 42.3% （11/26） of corresponding fecal samples, respectively. In contrast, no methylated SFRP2 gene was detected in mucosal tissues of normal controls, while two cases of matched fecal samples from normal controls were detected with hypermethylated SFRP2. A significant decrease （P 〈 0.001） in the rate of hypermethylated SFRP2 gene was detected in the postoperative （8.7%, 6/69） fecal samples as compared with the preoperative fecal samples （87%, 60/69） of CRC patients. Moreover, no significant associations were observed between SFRP2 hypermethylation and clinicopathological features including sex, age, tumor stage, site, lymph node status and histological grade, etc.CONCLUSION： Hypermethylation of SFRP2 gene in fecal DNA is a novel molecular biomarker of CRC and carries a high potential for the remote detection of CRC and premalignant lesions as noninvasive screening method.     

TFAP2E hypermethylation was associated with survival advantage in patients with colorectal cancer

Purpose

Hypermethylation of TFAP2E (AP-2E) is associated with the chemotherapy-resistant in patients with colorectal cancer (CRC), but its implications on prognosis directly remain unknown. This study was aimed to investigate the role of AP-2E methylation status and other clinicopathologic parameters as predictors of prognosis.

Methods

We detected the methylation status of AP-2E in tumor and adjacent non-tumor tissues from 311 sporadic CRC patients by methylation-sensitive high-resolution melting analysis. Log-rank tests and multivariate Cox analyses were performed to evaluate the role of AP-2E methylation status and other clinicopathologic parameters as predictors of prognosis.

Results

Hypermethylation of AP-2E was detected in 61 % (190/311) tumor tissues. It occurred more frequently in tumors in earlier stages (I/II; P = 0.02), lower levels of tumor invasion (T1–T3; P = 0.04), fewer lymph nodes involved (N0; P < 0.01), and higher histologic grades (G1/G2; P < 0.01). The overall 5-year survival rates in hypermethylation and hypomethylation group were 76.91 and 47.17 % (P < 0.0001), respectively. AP-2E hypermethylation was significantly associated with a favorable clinical outcome with a hazard ratio of 0.486 (95 % CI 0.342–0.692, P < 0.0001) after controlling for age, gender, tumor location, histologic type, TNM staging, and histologic grade.

Conclusions

AP-2E was frequently hypermethylated in tumors from patients with CRC. Aberrant hypermethylation of AP-2E occurred more frequently in tumors with earlier stages, lower levels of tumor invasion, fewer lymph nodes involved, and higher histologic grades. AP-2E hypermethylation might be an independent predictor of survival advantage in patients with CRC.     

EZH2和VEGF在结直肠癌中的表达其与患者预后关系生物信学分析及验证

背景结直肠癌(colorectal carcinoma, CRC)是临床上常见的消化系统恶性肿瘤.但其确切发病机制和预后独立因素仍未阐明.本研究通过生物信息学方法分析了zeste基因增强子同源物2(enhancerofzestehomolog2,EZH2)和血管内皮生长因子(vascular endothelial growth factor, VEGF)基因在CRC中的表达情况及其和患者预后的感谢.并采用免疫组化检测了EZHE2和VEGF蛋白的表达情况及其与患者临床特征的关系.目的探讨EZH2和VEGF在CRC中的表达、突变情况及其与患者临床病理特征和预后的关系.方法首先在TCGA数据库中比较EZH2和VEGF基因mRNA在肠癌患者癌和癌旁组织中的表达,同时分析EZH2和VEGF基因的突变情况;采用STRING数据库建立EZH2和VEGF基因表达网络并筛选网络中的关键基因.根据EZH2和VEGF在肿瘤组织中的表达分为高低表达组, Cox回归模型Log-rank检验比较高低表达组患者总生存和无疾病进展生存是否存在差异.同时选取80例肠癌手术患者,留取患者癌组织和癌旁组织,采用免疫组织化学法检测上述组织中EZH2和VEGF蛋白表达水平.结果 TCGA数据库显示EZH2和VEGF基因在CRC组织中的表达水平显著高于对应的正常肠上皮组织(P <0.05),而与肠癌患者的临床分期并无明显相关性(P>0.05);EZH2和VEGF基因在人肠癌中的突变率分别为1.5%和1.9%,且EZH2和VEGF不同突变组织中mRNA表达水平存在明显差异.网络中共有22个蛋白,各蛋白平均相互作用指数为10.5,区域聚集指数为0.8,各蛋白富集明显(P<0.01). Cytohubb软件筛选出EZH2, DNMT1, HDAC2, YY1和SUZ12为网络中的关键基因;EZH2和VEGF高低表达与患者总生存期均无相关性(P>0.05),而VEGF高表达组患者无疾病进展生存期显著低于低表达组(HR=1.8, P<0.05).免疫组化显示, EZH2和VEGF在肠癌组织中的阳性表达率均显著高于癌旁组织(P<0.01).EZH2阳性表达与肠癌肿瘤直径、分化程度和Duke分期有关(P<0.05).而VEGF阳性表达与肠癌患者分化程度和Duke分期存在相关性(P<0.05).结论 EZH2和VEGF在肠癌组织中呈现明显上调表达和突变,其高表达与肿瘤大小、分会程度和Duke分期有关,并可作为肠癌预后的潜在分子标志物.     

胃癌血流灌注状态与血清血管新生指标的相关性

背景准确评估肿瘤组织的微循环血流灌注情况,对于临床治疗以及预后评估有着重要意义.超声造影(contrast-enhanced ultrasonography,CEUS)是一种敏感度极高的微循环血流监测手段,能客观反映肿瘤组织的微循环血流灌注状态,可为临床诊治提供可靠的血流动力学信息.目的运用CEUS评估胃癌(gastric cancer,GC)血流灌注状态,并探讨其与血管新生指标的相关性.方法选取在浙江医院行手术切除的66例GC患者作为研究对象(GC组).所有患者术前三天内行CEUS检查,运用时间-强度曲线测定GC组织、癌旁正常组织的增强强度、达峰时间,并采用酶联免疫吸附法(enzymelinked immunosorbent assay,ELISA)测定患者血清血管内皮生长因子(vascular endothelial growth factor,VEGF)、血管生成素-2(Angiopoietin-2,Ang-2)水平,术后测定组织标本微血管密度(microvessel density,MVD).同期选取72例健康志愿者作为对照组进行比较分析.结果GC组织的增强强度明显高于癌旁正常组织,差异有统计学意义(P<0.05);GC组织的达峰时间明显短于癌旁正常组织,差异有统计学意义(P<0.05);GC组织的MVD明显高于癌旁正常组织,差异有统计学意义(P<0.05);GC组的VEGF、Ang-2明显高于对照组,差异有统计学意义(P<0.05);GC组织的增强强度分别与VEGF、Ang-2、MVD呈正相关(r=0.85,r=0.81,r=0.88,P<0.05);GC组织的达峰时间分别与VEGF、Ang-2、MVD呈负相关(r=-0.72,r=-0.73,r=-0.86,P<0.05).结论CEUS能动态评估GC血流灌注状态,其血流参数与VEGF、Ang-2、MVD相关性良好,能为临床评估GC血管新生状态提供无创性影像学手段.     

Usefulness of plasma epigenetic changes of five major genes involved in the pathogenesis of colorectal cancer

Purpose

The purpose of present study was to investigate the methylation status of the promoter region in five genes (mothers against decapentaplegic homolog 4, fragile histidine triad protein, death-associated protein kinase 1, adenomatous polyposis coli (APC), and E-cadherin), which are known to be involved in the pathogenesis of colorectal cancer (CRC) and its clinicopathological significance.

Methods

The study subjects were 60 CRC patients, 40 patients with adenomatous colorectal polyp and 60 healthy control individuals. We further enrolled a total of 16 patients (two patients with Crohn’s disease, two patients with ulcerative colitis, one patient with serrated adenoma, and 11 patients with colorectal cancer). The methylation states of the five genes were determined in peripheral blood plasma using methylation-specific polymerase chain reaction single-strand conformation polymorphism analysis.

Results

This study showed the most sensitive epigenetic markers, E-cadherin (60 %), followed by APC (57 %), for detecting CRC. E-cadherin and APC had similar specificities and amplified 84 and 86 %, respectively, of CRC patients compared to non-CRC patients. Additionally, APC was the only marker to be significantly increased (OR?=?6.67, 95 % CI?=?1.19–23.4, P?=?0.045) and the most sensitive (57 %) and specific (89 %) marker in stage I CRC. Though we have not examined the paired cancer tissues and plasma, there was relatively high concordant rate (60–80 %) in our limited number of colorectal cancer patients.

Conclusions

Five genes, promoter methylation, in plasma were statistically significant risk factors in CRC patients. In this study, E-cad and APC genes may be particularly useful epigenetic biomarkers in plasma for the detection of CRC. Additionally, APC may able to identify early potential CRC.     

Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

Background

CpG island hypermethylation of tumor suppressor genes is highly involved in gastric carcinogenesis, and enhanced cell proliferation could accelerate this process. Cyclin D1 regulates cell cycle function and may play a role in methylation-related carcinogenesis.

Aims

We investigated the association between Cyclin D1 gene G870A polymorphism and the methylation status of tumor suppressor genes in gastric cancer.

Methods

Polymorphisms at G870A in the Cyclin D1 gene were genotyped, and methylation status of the p14, p16, DAP-kinase, and CDH1 genes were determined by methylation-specific-polymerase chain reaction in 139 gastric cancer tissues. CIHM high was defined as three or more methylated CpG islands.

Results

Although no association was found between methylation status and different stages and Lauren’s subtypes, patients with CIHM of DAP-kinase showed significantly worse survival than those without (p = 0.017). In addition, the number of methylated sites was also associated with survival curves (p = 0.0397). The 870G carrier a significantly lower prevalence of CIHM high compared to the AA genotype in advanced-stage gastric cancer (adjusted OR = 0.32, p = 0.047). A weak correlation between the same genotypes and CIHM of p14 were found in the same subtype (adjusted OR = 0.32, p = 0.052). The mean methylation number was significantly lower in G carriers than in AA genotypes in advanced-stage gastric cancer (p = 0.017).

Conclusions

Genetic polymorphism of CCND1 is associated with CIHM status in gastric cancer, especially in the advanced stage, but is independent of clinico-pathological features.     

Fibulin-1 Is Downregulated Through Promoter Hypermethylation in Colorectal Cancer: A Consort Study

Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in colorectal cancer (CRC) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CRC.The expression of FBLN1 in CRC tissues and adjacent normal tissues was analyzed by immunohistochemical analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) were performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics.Immunohistochemical analysis and qRT-PCR analysis showed that FBLN1 protein and messenger RNA (mRNA) levels in tumor tissues were both significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001). In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant.Our results indicated that the FBLN1 gene may be a novel candidate of tumor suppressor gene in CRC, and that promoter hypermethylation of FBLN1 is an important reason for its downregulation and is also a good predictor of OS for CRC.     

New trends in molecular and cellular biomarker discovery for colorectal cancer

Colorectal cancer(CRC) is the third leading cause of cancer death worldwide, which is consequence of multistep tumorigenesis of several genetic and epigenetic events. Since CRC is mostly asymptomatic until it progresses to advanced stages, the early detection using effective screening approaches, selection of appropriate therapeutic strategies and efficient follow-up programs are essential to reduce CRC mortalities. Biomarker discovery for CRC based on the personalized genotype and clinical information could facilitate the classification of patients with certain types and stages of cancer to tailor preventive and therapeutic approaches. These cancer-related biomarkers should be highly sensitive and specific in a wide range of specimen(s)(including tumor tissues, patients' fluids or stool). Reliable biomarkers which enable the early detection of CRC, can improve early diagnosis, prognosis, treatment response prediction, and recurrence risk. Advances in our understanding of the natural history of CRC have led to the development of different CRC associated molecular and cellular biomarkers. This review highlights the new trends and approaches in CRC biomarker discovery, which could be potentially used for early diagnosis, development of new therapeutic approaches and follow-up of patients.     

LncRNA MEG3 acts a biomarker and regulates cell functions by targeting ADAR1 in colorectal cancer

BACKGROUND Colorectal cancer (CRC) is the third most prevalent malignancy and has the fourth highest global cancer mortality rate. Early diagnosis and prompt medical attention can improve quality of life and the prognosis of CRC patients. Accumulating evidence reveals that long non-coding RNAs (lncRNAs) function as oncogenes or anti-oncogenes, as well as biomarkers in various cancers. AIM To investigate the levels and molecular mechanism of the lncRNA maternally expressed gene 3 (MEG3) in CRC. METHODS The levels of lncRNA MEG3 in CRC tissue, serum and cell line samples were explored via qRT-PCR. The relationship between MEG3 levels and clinicopathological features in CRC was investigated. The diagnostic and prognostic values of serum MEG3 levels were analyzed with ROC curves and KaplanMeier survival curves, respectively. RESULTS Significant decreased levels of MEG3 existed in CRC tissue, cell lines and serum. CRC patients with down-regulated serum MEG3 levels had larger tumor sizes, and advanced clinical stages. The sensitivity and specificity of serum MEG3 levels in CRC detection was 0.667 and 0.875, respectively. Tumor size, T stages, and serum MEG3 levels are indie factors that produce an effect on CRC patients' prognosis. KaplanMeier survival curves suggested that CRC patients with high levels of MEG3 had a remarkably better overall survival rate. CONCLUSION LncRNA MEG3 is down-regulated in CRC, and regulates cell functions by targeting adenosine deaminase’s effect on RNA 1 in CRC.     

食管鳞癌组织p16基因调控区甲基化及其蛋白表达研究

目的探讨p16基因在食管癌变过程中表达缺失与其启动子区甲基化的关系。方法采用MSP免疫组化方法,检测环太行山地区45例食管鳞癌患者癌组织p16基因启动子区甲基化状态及蛋白表达情况。结果p16基因在癌组织中表达异常41例（91．1％）,间变组织中表达异常38例（84．4％）,发生纯合型甲基化的组织分别为33例（73．3％）（癌组织）和32例（71．1％）（间变组织）,而其周围正常组织26例（57．8％）均发生了p16启动子区的杂合型甲基化。p16基因纯合型甲基化与癌组织、间变组织、p16蛋白表达缺失相关（P〈0．05）。结论该地区食管癌组织p16基因在癌前病变中p16启动子区即发生了纯合型甲基化、食管癌变的早期事件。p16基因启动子区甲基化可单独影响p16蛋白的正常表达。     

人粪便SFRP2基因甲基化分析对结直肠癌的诊断价值

目的探讨人粪便中分泌型卷曲相关蛋白2(SFRP2)基因甲基化分析用于结直肠癌(CRC)早期诊断的可行性。方法从87例结直肠癌或良性病变的患者及24例正常对照者的粪便中分别提取DNA,采用甲基化特异性PCR(MSP)技术分析其SFRP2基因甲基化状态。结果CRC、腺瘤、增生性息肉和溃疡性结肠炎患者的SFRP2基因甲基化阳性率分别为94.2%(49/52)、52.4%(11/21)、37.5%(3/8)和16.7%(1/6)。1例正常对照SFRP2基因甲基化检测阳性。检测SFRP2基因甲基化诊断CRC及癌前病变的敏感性和特异性分别为90.5%和85.4%。结论SFRP2基因甲基化是CRC进展过程中的早期事件。粪便SFRP2基因甲基化分析可望成为CRC早期无创诊断或CRC高风险人群筛查的新途径。