Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients

AIM: To evaluate the diagnostic role of serum RASSF1A promoter hypermethylation in gastric and colorectal adenocarcinoma.METHODS: Methylation-specific polymerase chain reaction (MSPCR) was used to examine the promoter methylation status of the serum RASSF1A gene in 47 gastric adenocarcinoma patients, 45 colorectal adenocarcinoma patients, 60 patients with benign gastrointestinal disease (30 with benign gastric disease and 30 with benign colorectal disease), and 30 healthy donor controls. A paired study of RASSF1A promoter methylation status in primary tumor, adjacent normal tissue, and postoperative serum were conducted in 25 gastric and colorectal adenocarcinoma patients who later were underwent surgical therapy.RESULTS: The frequencies of detection of serum RASSF1A promoter hypermethylation in gastric (34.0%)and colorectal (28.9%) adenocarcinoma patients were significantly higher than those in patients with benign gastric (3.3%) or colorectal (6.7%) disease or in healthy donors (0%) (P ＜ 0.01). The methylation status of RASSF1A promoter in serum samples was consistent with that in paired primary tumors, and the MSPCR results for RASSF1A promoter methylation status in paired preoperative samples were consistent with those in postoperative serum samples. The serum RASSF1A promoter hypermethylation did not correlate with patient sex, age, tumor differentiation grade, surgical therapy,or serum carcinoembryonic antigen level. Although the serum RASSF1A promoter hypermethylation frequency tended to be higher in patients with distant metastases,there was no correlation between methylation status and metastasis.CONCLUSION: Aberrant CpG island methylation within the promoter region of RASSF1A is a promising biomarker for gastric and colorectal cancer.     

肺癌患者Ras相关区域家族1A基因启动子异常甲基化的检测

目的探讨肺癌组织和外周血浆、支气管肺泡灌洗液(BALF)中Ras相关区域家族1A(RASSF1A)基因启动子异常甲基化状况及其在肺癌诊断中的价值。方法用甲基化特异PCR方法对肺癌患者癌组织、癌旁组织及相应血浆、BALF进行RASSF1A异常甲基化检测。结果45例肺癌组织中,RASSF1A基因启动子异常甲基化率为53.33%(24/45),相应血浆中RASSF1A的甲基化检出率为28.89%(13/45),BALF检出率为42.22%(19/45),而癌旁组织中的RASSF1A启动子甲基化检出率为13.04%(3/23)、正常对照血浆、非肺癌患者BALF中未检出甲基化,只检出未甲基化的RASSF1A。血浆、BALF中甲基化改变与肿瘤组织甲基化状况显著相关(P<0.01);但与患者年龄、性别、肿瘤大小、恶性程度、肿瘤分类的差异无统计学意义(P>0.05)。结论血浆、BALF中RASSF1A基因异常甲基化改变的检测在肺癌的特异诊断等方面有一定的应用价值。     

肝癌患者血液RASSF1A基因甲基化的检测及其临床意义

目的:探讨原发性肝细胞癌(HCC)患者血清中RASSF1A甲基化状况及RASSF1A甲基化作为一种新的肿瘤分子标志物在HCC早期无创性诊断中的意义和价值.方法:应用甲基化特异性PCR(MSP)技术检测35例HCC患者血清和10例健康对照血清中RASSF1A启动子区甲基化状况.结果:35例HCC患者血清中RASSF1A启动子区甲基化阳性率为40%, 10份健康对照血清中未出现RASSF1A基因甲基化. RASSF1A基因甲基化与HCC患者性别、伴肝硬化、乙肝表面抗原、甲胎蛋白、肿瘤大小、有无包膜、有无门静脉癌栓及病理分级等临床病理参数无关.结论:RASSF1A基因甲基化在HCC的发生中起重要作用, RASSF1A基因甲基化可能是HCC新的肿瘤分子标志物.     

High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma.

PURPOSE: To determine the presence of ras association domain family 1A (RASSF1A) promoter methylation in the tumor tissues and plasma of patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Methylation-specific polymerase chain reaction was used to detect RASSF1A methylation in DNA extracted from HCC tumors and paired plasma samples of 40 patients. The association of RASSF1A hypermethylation in tumor and plasma DNA of HCC patients with clinicopathological characteristics was also analyzed. RESULTS: RASSF1A promoter hypermethylation was detected in 37 of the 40 HCC tissues (92.5%). Of the paired plasma from the 40 HCC patients, aberrant methylation was detected in 17 (42.5%). No RASSF1A methylation was detected in the plasma in the absence of methylation in the corresponding tumor. The presence of RASSF1A promoter hypermethylation in plasma DNA was found to associate with HCC size of > or =4 cm (P = 0.035). CONCLUSION: RASSF1A promoter hypermethylation occurred at a high frequency in HCC. The aberrant methylation was also detectable in over 40% of matched plasma. The latter should be evaluated as a screening tool and/or prognosticator of HCC patients.     

RUNX3、RASSF1A启动子高甲基化与胃癌进展转移的关系

目的:探讨胃癌RUNX3、RASSF1A基因启动子甲基化在胃癌进展转移中的作用及意义.方法:RT-PCR和MSP检测62例胃癌标本及56例正常胃黏膜组织RUNX3、RASSF1A基因mRNA表达及甲基化状况,免疫组织化学检测VEGF在RUNX3、RASSF1A甲基化与非甲基化胃癌组织及20例正常组织中的表达,并分析RUNX3、RASSF1A甲基化与VEGF表达的关系.结果:胃癌组织RUNX3与RASSF1A表达较正常组织均明显降低(0.629±0.461 vs 0.893±0.543,0.653±0.476 vs 0.858±0.581,均P<0.05),且RUNX3与RASSF1A甲基化率均高于正常组织(69.4%VS 26.8%,66.1%vs 23.2%.均P<0.01).胃癌组织中RUNX3、RASSF1A甲基化组mRNA表达量较非甲基化组明显降低(0.545±0.299 vs 0.736±0.291,0.562±0.208 vs 0.674±0.185,均P<0.05).RASSF1A甲基化与肿瘤TNM分期及浸润深度相走RUNX3甲基化与肿瘤淋巴结转移、血管侵犯及TNM分期相关(P<0.05).RUNX3甲基化组胃癌组织VEGF蛋白表达高于非甲基化组(86.0%vs57.9%),RUNX3甲基化与VEGF表达相关(P<0.05).结论:RUNX3、RASSF1A启动子高甲基化可能是导致其表达降低的原因,并与胃癌进展演变相关.RUNX3甲基化可能参与胃癌血管、淋巴管转移.     

胃癌组织RASSF1基因启动子区甲基化的意义

目的:探讨抑癌基因RASSF1A启动子在胃癌组织及胃良性病变中甲基化的发生情况及与临床特征的关系.方法:采用甲基化特异性聚合酶链反应(MS- PCR)方法检测32例胃癌组织及32例相应的癌旁组织,以及46例胃良性病变中RASSF1A基因甲基化发生情况.结果:在32例胃癌DNA标本中,RASSF1A基因甲基化发生率为62.5%(20/32).而在32例癌旁组织中,只有1例存在甲基化,占3.1%(1/32),二者之间比较差异有统计学意义(P<0.05).在胃良性病变,浅表性胃炎和萎缩性胃炎中甲基化发生频率分别为3.3%和37.5%.RASSF1A基因甲基化发生率与胃癌分化程度、肿瘤大小及淋巴结转移之间无显著相关性.结论:RASSF1A基因启动子区甲基化在胃癌的发生发展中起重要作用.     

TIMP-3和RASSF1A在大肠癌中的表达及临床意义

目的:探讨TIMP-3和RASSF1A在大肠癌发生发展中的作用及与临床病理特征之间的关系,并且研究TIMP-3和RASSF1A之间的相关性.方法:应用免疫组织化学法检测大肠癌组织,大肠癌旁组织及正常大肠黏膜组织中TIMP-3和RASSF1A蛋白的表达量并结合患者的年龄、性别、分化程度、淋巴结转移等临床病理因素进行综合分析.采用Spearman等级相关分析TIMP-3和RASSF1A之间的相关性.结果:在正常大肠黏膜、癌旁和腺癌中TIMP-3的阳性率分别为90.0%、70.0%和16.7%,RASSF1A阳性率分别为83.3%、63.3%和23.3%.大肠癌中TIMP-3和RASSF1A的表达量与淋巴结转移(P<0.05;P<0.01)、浸润深度(P<0.05;P<0.01)及分化程度(P<0.05;P<0.05)有关.运用Spearman等级相关分析,TIMP-3和RASSF1A的表达呈正相关(r=0.256,P<0.05).结论:在大肠癌中TIMP-3和RASSF1A具有明显相关性,TIMP-3和RASSF1A的表达下调可能与大肠癌的发生有关,可以作为鉴别大肠良恶性肿瘤的指标.     

胃癌组织RASSF1A甲基化对其mRNA及蛋白表达的影响

目的研究RASSF1A基因启动子区甲基化对胃癌组织中RASSF1AmRNA及蛋白表达的影响。方法RT-PCR方法检测54例胃癌及癌旁正常组织RASSF1AmRNA表达,甲基化特异性PCR方法检测RASSF1A启动子区CpG岛甲基化状态;Westernblot方法检测20例胃癌及癌旁正常组织中RASSF1A蛋白表达。结果(1)RASSF1AmRNA和蛋白表达水平在胃癌组织中明显低于癌旁正常组织(A值分别为0·2589±0·2407比0·5448±0·2971,P<0·0001;0·1874±0·0737比0·6654±0·2201,P<0·0001);(2)RASSF1A在胃癌组织和正常组织中的甲基化频率分别为66·7%和14·8%,差异有统计学意义(P<0·0001);(3)在胃癌组织中,甲基化组RASSF1AmRNA的表达明显低于非甲基化组(0·1384±0·1142比0·5018±0·2463,P<0·0001)。结论胃癌组织RASSF1AmRNA和蛋白表达缺失或低下,与其启动子甲基化程度增高显著相关。     

RASSF1A gene promoter methylation in esophageal cancer specimens

SUMMARY. RASSF1A is frequently inactivated by promoter methylation in human cancers. To understand the involvement of the RASSF1A gene in esophageal squamous cell cancer (ESCC), we investigated the methylation of the RASSF1A gene in primary ESCC to define the frequency of this epigenetic aberration and its clinicopathological significance. Methylation-specific polymerase chain reaction (MSP) was used to detect RASSF1A gene methylation in DNA from 55 cases of ESCC. Methylation of the RASSF1A gene was found in 13 of 55 (24%) cases of primary ESCC. No association was found between the promoter methylation of the RASSF1A gene in primary ESCC and age, gender, localization, invasion depth, or tumor stage. Association was found with tumor differentiation. There was no correlation with its prognosis. In conclusion, it was suggested that an inactivation of the RASSF1A gene due to promoter methylation was associated with de-differentiation of the tumor in ESCC.     

RASSF1A基因启动子区甲基化在贲门腺癌、食管下段鳞癌组织中的变化及临床意义

目的:比较RASSF1A基因甲基化在贲门腺癌、食管下段鳞癌不同病理阶段组织中的共性和差异.方法:33例贲门腺癌和36例食管下段鳞癌手术切除标本纳入本研究,每例标本选取癌组织,癌旁不典型增生组织(距癌3-5 cm),癌旁正常组织(距癌>5 cm)组织各1块,采用甲基化特异性PCR检测RASSF1A基因启动子区的甲基化情况...     

RASSF1A基因在胃腺癌和癌前病变中的表达及意义

目的: 探讨RASSF1A与CyclinD1在胃癌发生发展中的作用.方法: 采用RT-PCR检测胃正常组织、腺瘤组织、不典型增生组织各20例及胃腺癌组织40例中RASSF1A及CyclinD1 mRNAA表达,并Western blot法检测RASSF1A蛋白表达.结果: 胃腺癌组RASSF1A的表达低于不典型增生、良性腺瘤及正常组织组(37.5%vs 80.0%,95.0%,100.0%,均P<0.05),而CyclinD1的表达高于不典型增生、良性腺瘤及正常组织组(77.5%vs 25.0%,10.0%,5.0%,均P<0.05).胃癌组织中,RASSF1A及CyclinD1 mRNA表达均与病理分级有关(χ2=4.422,8.935,均P<0.05);两者之间表达呈负相关(γ=-0.448,P<0.05);RASSF1A蛋白表达与mRNA表达一致.结论: RASSF1A与CyclinD1两种蛋白的异常表达在胃癌的发生发展中可能具有协同作用,二者联合检测能为胃癌的早期临床诊断和治疗提供有利的生物学信息.     

胃癌RASSF1A基因启动子CpG岛甲基化与DNMT1表达的意义

目的探讨RAS相关区域家族1A（RASSF1A）基因启动子CpG岛甲基化和DNA甲基转移酶1（DN-MT1）的表达与胃癌发生的关系。方法运用甲基化特异性聚合酶链反应和免疫组织化学方法检测胃癌癌旁正常组织和癌组织RASSF1A基因启动子CpG岛甲基化发生率及DNMT1的表达情况。结果癌旁正常组织中RASSF1A基因启动子CpG岛甲基化发生率、DNMT1阳性表达率显著低于相应癌组织（P均〈0.01）。RASSF1A启动子CpG岛甲基化患者DNMT1阳性表达率与非甲基化患者比较无统计学差异（P〉0.05）。结论RASSF1A基因启动子区CpG岛甲基化和DNMT1的高表达可能与胃癌发生有一定关系。     

Activator protein-1 involved in growth inhibition by RASSF1A gene in the human gastric carcinoma cell line SGC7901

AIM：To investigate the role of Ras association domain family protein 1 isoform A （RASSF1A） in gastric tumorigenesis. METHODS：Through over-expression of RASSF1A gene in the SGC7901 cell line which was induced by a lipofectamine-mediated gene transfer approach. Activator protein-1 （AP-1） DNA binding activity was measured by electrophoretic mobility shift assay （EMSA）. RESULTS：Compared with the control clones, cells over- expressing RASSF1A exhibited significant inhibition of cell growth with G1 cell cycle arrest in vitro and in vivo. The over-expression of RASSF1A significantly inhibited AP-1 activity in SGC7901 cells （0.981±0.011 vs 0.354±0.053, P〈0.001）. In addition, both Western blot analysis and immunocytochemistry demonstrated that RASSF1A down-regulated the expression of c-Fos （0.975± 0.02 vs 0.095±0.024, P〈0.001） but not c-Jun.
CONCLUSION： Over-expression of RASSF1A inhibits the growth of SGC7901 cells by negatively regulating the AP-1 activity, the latter in turn negatively signals cell proliferation.     

Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma

Purpose  

Protein downregulation and hypermethylation of Ras association domain family 1A (RASSF1A) has been recognized as an important early event in different classes of carcinogenesis, but clinicopathological significance of RASSF1A protein expression and methylation in hepatocellular carcinoma (HCC) remains largely unknown. The aim of the study was to investigate the expression of RASSF1A protein and methylation in HCC and their clinical significance.     

Microsatellite instability and MLH1 promoter hypermethylation in colorectal cancer

Colorectal cancer (CRC) is caused by a series of genetic or epigenetic changes, and in the last decade there has been an increased awareness that there are multiple forms of colorectal cancer that develop through different pathways. Microsatellite instability is involved in the genesis of about 15% of sporadic colorectal cancers and most of hereditary nonpolyposis cancers. Tumors with a high frequency of microsatellite instability tend to be diploid, to possess a mucinous histology, and to have a surrounding lymphoid reaction. They are more prevalent in the proximal colon and have a fast pass from polyp to cancer. Nevertheless, they are associated with longer survival than stage-matched tumors with microsatellite stability. Resistance of colorectal cancers with a high frequency of microsatellite instability to 5-fluorouracil-based chemotherapy is well established. Silencing the MLH1 gene expression by its promoter methylation stops the formation of MLH1 protein, and prevents the normal activation of the DNA repair gene. This is an important cause for genomic instability and cell proliferation to the point of colorectal cancer formation. Better knowledge of this process will have a huge impact on colorectal cancer management, prevention, treatment and prognosis.     

XRCC1 downregulated through promoter hypermethylation is involved in human gastric carcinogenesis

OBJECTIVE: To analyze the expression and aberrant methylation of X-ray repair cross-complementing gene 1 (XRCC1) in gastric carcinogenesis, and identify the molecular mechanism of gastric carcinogenesis. METHODS: The method based on methyl binding domain protein (MBD) immuno-precipitation and promoter microarray was employed to screen the gastric cancer-related methylation-sensitive gene. An immunohistochemistry assay was applied to detect the protein expression of XRCC1 in the multistep progression of gastric carcinogenesis. The mRNA expression of XRCC1 was determined by real-time PCR in tumor tissues and their corresponding non-tumorous tissues. The methylation status and Arg194Trp and Arg399Gln polymorphisms of XRCC1 in gastric cancer and gastritis tissues were analyzed by methylation-specific PCR, bisulfite genomic sequencing and direct DNA sequencing, respectively. RESULTS: Promoter microarray screening and identification suggested that XRCC1 was a methylation-sensitive gene. Immunochemistry results showed that XRCC1 protein expression gradually decreased with progression of gastric mucosal lesions (P < 0.05). The positive rate of XRCC1 in patients with well/moderately differentiated gastric cancer was significantly higher than patients with poorly differentiated gastric cancer (P < 0.05). The mRNA expression of XRCC1 in gastric cancer tissues was significantly lower than that in the non-tumorous tissues (P < 0.05). Meanwhile, XRCC1 methylation in gastric cancer tissues was more frequent than that in the gastritis tissues (P < 0.05), and the downregulation of XRCC1 expression was relevant to methylation (P < 0.05). CONCLUSION: The expression of XRCC1 is downregulated in gastric carcinogenesis, and promoter hypermethylation may be one of the mechanisms contributing to its downregulation.     

广西肺鳞状细胞癌RASSF1A及RPRM基因甲基化分析

目的研究RASSF1A及RPRM甲基化在广西肺鳞状细胞癌患者中的发生频率。方法采用甲基化特异性PCR（MSP）方法检测30例临床上经病理确诊为肺鳞状细胞癌患者（肺癌组）和20例良性肺部疾病患者（对照组）RASSF1A及RPRM甲基化的状态,建立广西肺鳞状细胞癌相关基因的甲基化谱式。结果肺癌组RASSF1A及RPRM甲基化率分别为83.3%（25/30）和70.0%（21/30）,对照组分别为0和10%（2/20）,P均〈0.01。结论RASSF1A及RPRM基因DNA甲基化状态的检测有助于准确诊断肺鳞状细胞癌。     

Methylated APC and RASSF1A in multiple specimens contribute to the differential diagnosis of patients with undetermined solitary pulmonary nodules

Background

Inactivation of tumor-suppressor gene (TSG) by promoter hypermethylation has been reported in many tumor types, including lung cancer. This study was designed to determine the methylated APC and RASSF1A genes in tumor tissue, serum and plasma of patients with early stage lung cancer.

Methods

Eighty-nine patients with undetermined solitary pulmonary nodules detected upon CT-scan were recruited in this study. DNA samples were extracted from biopsy tissues, serum and plasma and QMSP of APC and RASSF1A was carried out after bisulfite conversion. The 89 patients consist of 58 stage I lung cancer patients and 31 benign lung disease according to pathological report. Twenty-six cancer patients had matched biopsy tumor tissue, serum and plasma samples.

Results

The methylation rates of APC and RASSF1A were 59.0% and 66.1% in biopsy tissues, 42.5% and 52.5% in serum, and 24.1% and 43.1% in plasma of cancer patients. For RASSF1A, different samples all showed a significant difference between cancer group and benign group (P<0.05). However, APC gene only explored the P value less than 0.05 in plasma result. Towards the 26 lung cancer patients with three matched samples, methylation rate in each sample type was more than 50.0% and displayed no difference.

Conclusions

Evaluation of APC and RASSF1A promoter methylation by using QMSP appears to be very useful for the differential diagnosis of patients with undetermined solitary pulmonary nodules. Our results also suggested that plasma might be the best sample for clinical detection of early stage lung.