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.     

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.     

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.     

Research on the reactivation of Syk expression caused by the inhibition of DNA promoter methylation in the lung cancer

The aim of this study was to study the expression of Syk gene and methylation in its promoter region in the lung cancer and to investigate the relationship between silencing of the Syk gene and DNA methylation of the Syk promoter region in lung cancer cell lines. Real-time PCR and immunohistochemistry were used to examine the Syk expression in specimens from 3 lung cancer cell lines and 16 lung cancer patients (tumor tissues and adjacent normal tissues). MSP was used to analyze the methylation status of the Syk promoter region. We also investigated the role of restoring Syk expression by using a DNA methyltransferase inhibitor, 5-aza-CdR, in suppressing invasion of lung cancer cell lines. No expression of the Syk gene was detected in the 3 lung cancer cell lines. In the 16 lung cancer patient samples, Syk expression was significantly lower in the tumor tissues than that in their adjacent normal tissues (P<0.05). Consistently, immunohistochemistry analysis of Syk protein expression showed that in the lung cancer tissues Syk protein expression was also significantly lower than that in their adjacent normal tissues. In the two lung cancer cell lines (NL9980, YTMLC-9) that lack the endogenous Syk expression, 4uM demethylation agent 5-aza-CdR treatment was able to reactivate the Syk gene expression, but not NCI-H446. In conclusion, hypermethylation leads to silencing of the Syk gene in human lung carcinoma cell lines. Methylation of the Syk promoter and loss of Syk expression in lung cancer cell lines are independent biomarkers. Syk may be a potential tumor suppressor in human lung 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。     

Helicobacter pylori induces promoter hypermethylation and downregulates gene expression of IRX1 transcription factor on human gastric mucosa

Background and Aim: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. Methods: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori‐positive chronic gastritis patients or H. pylori‐negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES‐1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES‐1 gastric mucosa cells. Results: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30% ± 13.17 versus 5.20% ± 6.31, respectively (P < 0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES‐1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. Conclusions: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.     

JAM3基因甲基化是食管鳞状细胞癌潜在的分子标志物

目的研究JAM3基因启动子区在食管癌中的甲基化情况及其表达调控机制,探讨JAM3基因启动子区异常甲基化作为食管鳞状细胞癌的潜在诊断标志物和治疗靶标。方法应用半定量RT-PCR、甲基化特异性PCR等技术对7个食管癌细胞系(KYSE140、KYSE150、KYSE410、KYSE450、COLO680N、KYSE520和TE13)、5例正常食管黏膜组织和83例原发性食管鳞状细胞癌组织进行分析。结果JAM3 mRNA在KYSE520、KYSE140、KYSE450细胞中高表达,这些细胞的JAM3基因启动子区呈非甲基化状态。JAM3 mRNA在KYSE410、COLO680N、TE13、KYSE150细胞中表达缺失,且其基因启动子区在这些细胞中呈完全甲基化。经过5-Aza-dc处理后,JAM3基因在KYSE410、COLO680N、TE13、KYSE150细胞中恢复表达。这些结果表明,JAM3在食管癌细胞中的表达受启动子区甲基化的调控。JAM3基因启动子区在5例正常食管黏膜组织中呈非甲基化状态(0/5),而在原发性食管鳞状细胞癌中其甲基化率为50.6%(42/83),且JAM3甲基化与肿瘤的位置相关(P<0.05)。结论JAM3在食管鳞状细胞癌中频繁发生甲基化,JAM3基因的表达受启动子区甲基化的调控,JAM3基因是潜在的食管癌诊断标志物和治疗靶标。     

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.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

Aim. Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. Method. A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). Results. Aberrant p16 promoter methylation was found in 64.3% (n=28) of ESCC family members and none (n=30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n=30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. Conclusion. Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.