IQGAP2抑制结肠癌细胞侵袭且通过启动子异常甲基化致基因沉默

目的 探讨在结肠癌细胞系中,含IQ模序GTP酶激活蛋白(IQGAP)2的启动子甲基化状态及IQGAP2对结肠癌细胞侵袭的影响.方法 在人结肠癌RKO细胞株中,采用实时荧光定量聚合酶链反应(RT-PCR)、脱甲基化试剂5-aza-2'-deoxycytidine处理,甲基化特异性聚合酶链反应(MSP)法、甲基化测序检测I...     

Downregulation of retinoic acid receptor-β2expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM:To study the role of hypermethylation in the loss of retinoic acid receptor β2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS:The role of hypermethylation in RARβ2 gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP),and its methylation status was compared with RARβ2 mRNA expression by RT-PCR.The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions.RESULTS:Methylation was detected in 4 of the 6 cell lines,and the expression of RARβ2 was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2 was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ2 after 5-aza-2′-deoxycytidine (5-aza-dc) treatment.CONCLUSION:The methylation of the 5′ region may play an important role in the downregulation of RARβ2 in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines.This study may have clinical applications for treatment and prevention of ESCC.     

Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues

INTRODUCTION Human tumors often display changes in DNA methylation, which include both genome-wide hypomethylation and site-specific hypermethylation. Global hypomethylation and CpG island hypermethylation have been recognized as important contributors to…     

结直肠癌组织及细胞株中分泌型卷曲相关蛋白家族的甲基化研究

目的 研究分泌型卷曲相关蛋白(SFRP)基因启动子区CpG岛甲基化与结直肠癌的关系.方法 采用甲基化特异性PCR(MSP)技术检测20对结直肠癌及相应癌旁组织中SFRP基因启动子区CpG岛甲基化状况,T-A克隆测序验证MSP产物.5-氮杂-2'-脱氧胞苷对结直肠癌细胞株HCT116、SW480进行去甲基化处理,MSP和Western印迹法分别检测细胞株中SFRP基因甲基化和蛋白表达.结果 SFRP 1、2、4、5在结直肠癌中甲基化率分别为19/20、17/20、3/20、13/20,癌旁组织中分别为12/20、8/12、1/20、7/20.结直肠癌中SFRP 1、2、5甲基化率均高于癌旁组织,差异有统计学意义(P<0.05).HCT116细胞株SFRP 1、2、4、5基因均发生甲基化,而SW480细胞株中仅SFRP1和SFRP2出现启动子甲基化.SFRP蛋白表达与启动子甲基化呈明显负相关,经5-氮杂-2'-脱氧胞苷处理后能有效恢复SFRP蛋白表达.结论 SFRP 1、2、5甲基化可能与结直肠癌的发生有关,SFRP基因甲基化与其表达失活密切相关.     

Differential expression of hDAB2IPA and hDAB2IPB in normal tissues and promoter methylation of hDAB2IPA in hepatocellular carcinoma

BACKGROUND/AIMS: hDAB2IP is a candidate tumor suppressor gene. We studied the expression of its two variants, hDAB2IPA and hDAB2IPB, in normal tissues, and the expression and methylation status of hDAB2IPA in hepatocellular carcinomas (HCC) and cell lines. METHODS: Conventional or real-time RT-PCR was performed in normal tissue samples, cell lines and HCC samples, and sequencing analysis and methylation-specific PCR in cell lines and HCC samples. RESULTS: hDAB2IPA was the predominant isoform, being expressed in the majority of tissues examined. The expression of hDAB2IPA was silenced or down-regulated but could be restored by 5-aza-2'-deoxycytidine treatment in liver cancer cell lines. The reactivation of hDAB2IPA was associated with promoter demethylation. The correlation between promoter methylation and hDAB2IPA expression was confirmed in eight pairs of matched HCC samples. Further, the methylation of the hDAB2IPA promoter in HCC was confirmed in an additional 53 pairs of patient samples. More than 80% of HCC samples showed hDAB2IPA promoter methylation, compared to 11.5% in the corresponding adjacent normal tissue (p<0.0001, chi2). CONCLUSIONS: Our data suggest that hDAB2IPA is the dominant isoform expressed in normal tissues. Its expression is suppressed in HCC, consistent with its role as a tumor suppressor gene, mainly by promoter methylation.     

Downregulation of retinoic acid receptor-β2expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines

AIM: To study the role of hypermethylation in the loss ofretinoic acid receptorβ2(RARβ2) in esophageal squamous cell carcinoma (ESCC).METHODS: The role of hypermethylation in RAR,β2 gene silencing in 6 ESCC cell lines was determined by methylationspecific PCR (MSP), and its methylation status was compared with RARβ2 mRNA expression by RT-PCR. The MSP results were confirmed by bisulfite sequencing of RARβ2promoter regions. RESULTS: Methylation was detected in 4 of the 6 cell lines, and the expression of RARβ2was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARβ2was restored in one RARβ2-downregulated cell line with the partial demethylation of promoter region of RARβ after 5aza-2'-deoxycytidine (5-aza-dc) treatment.CONCLUSION: The methylation of the 5' region may play an important role in the downregulation of RARβ2 in someESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARβ2expression in ESCC cell lines. This study may have clinical applications for treatment and prevention of ESCC.     

Transcriptional silencing of Dickkopf gene family by CpG island hypermethylation in human gastrointestinal cancer

AIM： To clarify alterations of Dickkopfs （Dkks） and Kremen2 （Krm2） in gastrointestinal cancer.
METHODS： We investigated the expression profiles and epigenetic alterations of Dkks and Krm2 genes in gastrointestinal cancer using RT-PCR, tissue microarray analysis, and methylation specific PCR （MSP）. Cancer cells were treated with the demethylating agent and/or histone deacetylase inhibitor. WST-8 assays and/n y/tro invasion assays after treatment with specific siRNA for those genes were performed.
RESULTS： Dkks and Krm2 expression levels were reduced in a certain subset of the gastrointestinal cancer cell lines and cancer tissues. This was correlated with promoter hypermethylation. There were significant correlations between Dkks over-expression levels and beta-catenin over-expression in colorectal cancer. In colorectal cancers with beta-catenin over-expression, Dkk-1 expression levels were significantly lower in those with lymph node metastases than in those without. Down-regulation of Dkks expression by siRNA resulted in a significant increase in cancer cell growth and invasiveness in vitro.
CONCLUSION： Down-regulation of the Dkks associated to promoter hypermethylation appears to be frequently involved in gastrointestinal tumorigenesis.     

5-氮杂-2′-脱氧胞苷对胃癌AGS细胞CHFR基因去甲基化作用及意义

目的观察5-氮杂-2′-脱氧胞苷（5-Aza-CdR）对胃癌AGS细胞CHFR基因去甲基化的作用,并探讨其临床意义。方法分别采用BSP和RT-PCR技术检测5-Aza-CdR处理前后胃癌AGS细胞CHFR基因启动子甲基化状态及其mRNA。结果 AGS细胞CHFR基因启动子在5-Aza-CdR处理前呈现高甲基化状态（甲基化率≥60%）,其mRNA表达完全缺失;5-Aza-CdR处理后则表现为低或无甲基化状态（甲基化率≤20%）,其mRNA表达恢复正常。结论 CHFR基因启动子在AGS细胞中呈高甲基化状态,5-Aza-CdR能显著逆转其CHFR基因异常甲基化,诱导CHFR基因表达,为胃癌的治疗提供新思路。     

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma

AIM To screen clinically relevant micro RNAs (mi RNAs) silenced by DNA methylation in human hepatocellular carcinoma(HCC).METHODS Knockdown of DNA methyltransferases (DNMTs) using si RNAs and mi RNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated mi RNA downregulation. Confirmation using individual quantitative real-time PCR (qR T-PCR) assays was thenperformed followed by DNA methylation quantification at the promoter of the mi RNA genes. Quantification of DNA methylation and mi RNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables.RESULTS mi RNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of mi R-23, mi R-25 and mi R-183. After q RT-PCR confirmation and Cp G island methylation quantification of these miR NAs in cell lines, further analysis in primary HCC specimens showed that hsa-mi R-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of mi R-183. In HCC patients, hypermethylation at hsami R-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes.CONCLUSION Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.     

胰腺癌细胞株HOXA7基因表达及其启动子区甲基化状态

目的 检测HOXA7 mRNA在胰腺癌细胞株中的表达及其启动子区的甲基化状态,探讨两者的相关性.方法 采用RT-PCR法检测人胰腺癌细胞株BxPC3、CFPAC1、PANC1和SW1990细胞的HOXA7 mRNA的表达水平.采用重亚硫酸盐测序PCR(bisulfite sequencing PCR,BSP)和结合重亚硫酸盐的限制性内切酶法(combined bisulfite restriction analysis,COBRA)检测启动子区域甲基化状态.应用去甲基化药物5-氮杂-2'-脱氧胞苷(5-aza-2-deoxycytidine,5-aza-dC)处理各细胞株,检测处理前后细胞HOXA7 mRNA表达和甲基化状态的变化.结果 胰腺癌细胞株BxPC3、CFPAC1和SW1990细胞均表达HOXA7 mRNA,而PANC1细胞不表达HOXA7 mRNA.CFPAC1、BxPC3、PANC1和SW1990中HOXA7启动子甲基化率分别为93.16%、90.65%、90.09%、52.30%,SW1990细胞的HOXA7甲基化率较其他三株细胞均明显降低(P值均＜0.01).经5-aza-dC处理后,PANC1细胞的HOXA7 mRNA重新表达,BxPC3的HOXA7 mRNA表达增强;而CFPAC1和SW1990细胞的HOXA7 mRNA在5-aza-dC处理前后无明显变化.结论 胰腺癌细胞株BxPC3和PANC1细胞的HOXA7mRNA表达与启动子区甲基化状态密切相关,而CFPAC 1和SW1990细胞两者无明显相关性.     

Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer

AIM： To identify the methylation of secreted frizzled-related protein 1 （SFRP1） in gastric cancer and to investigate the aberrant expression of SFRP1 and its correlation with the clinical pathological features of patients.
METHODS： We determined SFRP1 methylation and SFRP1 mRNA expression in 3 gastric cancer cell lines SGC-7901, BGC-823, HGC-27, from 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens by methylation-specific （MSP） PCR and RT-PCR respectively. Fisher＇s exact test was used to analyze the statistical association between clinical pathological data and aberrant expression of SFRP1.
RESULTS： In 3 cancer cell lines, BGC-823 and HGC-27 had methylated SFRP1 and lost SFRP1 mRNA expression. After treatment of BGC-823 and HGC-27 with the demethylating agent, 5-aza-2′-deoxycytidine, SFRP1 was re-expressed. In 52 primary gastric cancer specimens and matched tumor adjacent tissue specimens, hypermethylation of SFRP1 was detected in 23 （44%） and 8 （15%） specimens respectively （x^2= 10.34, P 〈 0.01）. Loss of SFRP1 expression was detected in 17（33%） and 6 （12%） specimens respectively （x^2= 6.75, P 〈 0.01）. There was a significant correlation between SFRP1 hypermethylation and SFRP1 expression loss. SFRP1 expression was also correlated significantly with tumor stage and lymph node status, but not with patient sex, age and histological type.
CONCLUSION： SFRP1 inactivation is a common and early event caused mainly by hypermethylation in gastric cancer. SFRP1 expression loss may be correlated with tumor metastasis in primary gastric cancer.     

胃癌维甲酸信号通路相关基因启动子区协同高甲基化的研究

背景：启动子区高甲基化与胃癌中多种抑癌基因表达沉默密切相关。目的：探讨维甲酸信号通路相关基因维甲酸受体B（RAR13）、细胞维生素A结合蛋白1（CRBP1）和他扎罗汀诱导基因1（TIG1）启动子区高甲基化与胃癌的关系。方法：以甲基化特异性聚合酶链反应（MSP）检测40例胃癌标本、10例正常胃黏膜标本和6株胃癌细胞株的RAR13、CRBPI和TIG1基因启动子区甲基化状态，分析i者甲基化状态的相关性及其与胃癌1晦床病理特征的关系。以逆转录聚合酶链反应（RT—PCR）检测胃癌细胞株RAR13、CRBP1和TIG1mRNA表达。结果：40例胃癌组织的RAR13、CRBPI和TIG1基因甲基化率分别为45．0％、32．5％和57．5％，10例正常胃黏膜组织均未检测到上述基因甲基化（P〈0．05）。胃癌组织中RAR13的甲基化状态与CRBP1和TIG1的甲基化状态显著相关（P〈0．05），但三者的甲基化状态与胃癌临床病理特征无相关性。启动子区高甲基化胃癌细胞株相应基因mRNA表达缺失或减弱。结论：胃癌组织常发生维甲酸信号通路相关基因RAR13、CRBP1和TIG1启动子区高甲基化，高甲基化可能是相应基因转录失活的重要原因。     

Methylation of TIMP3 in esophageal squamous cell carcinoma

AIM： To measure the frequency of DNA methylation of the tissue inhibitor of metalloproteinase 3 （TIMP3） promoter and relate this to any change of gene expression in esophageal squamous cell carcinoma in patients from a region of high incidence in China. METHODS： Cancer cell lines were treated with or without the demethylating reagent 5-aza-2′-deoxycytidine. Methylation of the TIMP3 promoter was assessed in three regions by melt curve analysis and its expression was assessed by real-time RT-PCR. Tumors and proximal resection margins were obtained from 64 patients with esophageal squamous cell carcinoma from a region of high incidence in China. Methylation was assessed by melt curve analysis and expression by immunohistochemistry.
RESULTS： Methylation in one of the three promoter regions assessed correlated with gene silencing in esophageal cell lines. A degree of methylation of TIMP3 was found in only four esophageal squamous cell carcinomas, and partial loss of TIMP3 protein expression in just one.
CONCLUSION： Methylation and loss of expression of TIMP3 occurs infrequently in esophageal squamous cell carcinoma in a region of high incidence in China.     

Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

AIM： TO screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line. METHODS： Methylated DNA sequences in genome were enriched with methylated CpG islands amplification （MCA） to undergo representational difference analysis （RDA）, with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR （MSP） and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer. Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent. RESULTS： Nineteen differentially methylated sequences were obtained and located at 5＇ end, exons, introns and 3＇ end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, as the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signalsexcept with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent. CONCLUSION： Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical methods. Significant