SOX30基因在结直肠癌中的表达与甲基化分析

目的:探讨新发现的抑癌基因SRY盒包含基因30(SOX30)在结直肠癌中的表达及甲基化变化。方法:用逆转录PCR(RT-PCR)检测结直肠癌细胞株(HCT8、HCT116和SW480)中SOX30 mRNA的表达,利用去甲基化药物5-Aza-CdR处理结直肠癌细胞株72 h后检测SOX30基因高甲基化与其表达调控的关系。应用甲基化特异性PCR(MSP)检测上述3株结直肠癌细胞株及54例结直肠肿瘤组织和10例癌旁组织SOX30基因的甲基化发生情况,通过硫化测序PCR(BSP)验证结直肠癌组织和癌旁组织SOX30基因的甲基化改变,并对甲基化情况与结直肠癌患者病理特征进行相关性分析。结果:SOX30 mRNA在上述3株结直肠癌细胞株中表达与对照组相比均较低,且均发生明显高甲基化;去甲基化药物处理上述3株细胞株后,SOX30 mRNA的表达明显得到恢复。SOX30基因在大部分结直肠癌组织中均呈现高甲基化状态,甲基化发生率为79.6%(43/54);而在相应的癌旁组织中甲基化检出率较低,仅为20%(2/10)。对结直肠癌患者中SOX30基因甲基化与其临床病理学资料进行相关性分析发现,SOX30基因高甲基化与患者的肿瘤病理分型显著相关(P=0.045),而与患者的性别、年龄、吸烟与否、TNM分期和Ducks分期无显著相关性(P均>0.05)。结论:SOX30基因在结直肠癌中发生明显高甲基化修饰改变,其基因表达水平明显下调,而且其甲基化修饰率与患者肿瘤病理分型相关,提示SOX30可能在结直肠癌的发生过程中起重要作用。     

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.     

Identification of GABRA1 and LAMA2 as new DNA methylation markers in colorectal cancer

Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29?genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.     

Promoter hypermethylation of the ADAM23 gene in colorectal cancer cell lines and cancer tissues

Promoter hypermethylation of the ADAM23 gene, which is normally involved in cell‐to‐cell and cell‐to matrix adhesion, has been reported in pancreatic, breast and brain cancer, and recently the role of this gene was examined in gastric cancer. In this study, we analyzed ADAM23 expression in colorectal cancer cell lines and examined its methylation by methylation‐specific PCR (MSP) and bisulfate‐modified DNA sequencing analysis. Methylated cells were treated with 5‐aza‐2′‐deoxycytidine to restore the ADAM23 expression. We then examined ADAM23 methylation status in colorectal cancer tissues and their corresponding normal tissues. We found that ADAM23 was aberrantly silenced or expressed at very low levels in 28 of the 32 (88%) colorectal cancer cell lines. MSP analysis showed that ADAM23 was methylated in 29 of 32 (91%) colorectal cancer cell lines and attenuated expression of ADAM23 was found to be related to hypermethylation in its promoter region. Moreover, the CpG dinucleotide methylation threshold of 70–90% was found to be required for complete silencing. In addition, when some cell lines without ADAM23 expression were treated with 5‐aza‐2′‐deoxycytidine, ADAM23 was reexpressed. In colorectal cancer tissues, the promoter region of ADAM23 was hypermethylated in 36 of 76 (47%). These results demonstrated that ADAM23 may be down‐regulated by aberrant promoter hypermethylation during the progression of colorectal cancer. © 2008 Wiley‐Liss, Inc.     

Hypermethylation and Clinicopathological Significance of RASAL1 Gene in Gastric Cancer

Background: Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1)is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development ofgastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signalingpathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 generemains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer.Materials and Methods: Using the methylation-specific polymerase chain reaction (MSP), the methylation statusof CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 ingastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was alsodetermined after treatment with a DNA methyltransferase inhibitor, 5-aza-2’-doexycytidine (5-Aza-CdR). RASactivity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstreammolecule of RAS signaling pathways, were determined by Western blotting. Results: The frequencies of RASAL1promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30%(12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation withtumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer(all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 genewas detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell lineGES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detectedin three gastric cancer cell lines after treatment with 5-Aza-CdR. Conclusions: Aberrant hypermethylation ofthe RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylatingagent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have acertain relationship with the reduced RASAL1 expression in gastric cancer.     

A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer

Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

Frequent inactivation of SPARC by promoter hypermethylation in colon cancers

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.     

人脑胶质瘤细胞株中RIZ1基因启动子甲基化状态的研究

检测4种人脑成胶质细胞瘤细胞株中成视网膜细胞瘤蛋白结合锌指结构基因1（retinoblastoma protein-interacting zinc finger gene1,RIZ1）基因启动子区的甲基化状态,进一步认识RIZ1在胶质瘤发病机制中的作用。方法：应用甲基化特异性PCR法（methylation specific PCR,MSP）检测4种人脑成胶质细胞瘤细胞株U87、U251、A172和T98中RIZ1基因启动子区的甲基化水平,其中U87细胞发生甲基化,被选为后续实验对象。RT-PCR检测U87细胞经5-Aza-CdR处理前后RIZ1 mRNA表达量的变化,MTT检测5-Aza-CdR对U87细胞生长增殖的影响。结果：4种人脑成胶质细胞瘤细胞株中U87和U251细胞中检测到RIZ1基因启动子区域发生甲基化;U87细胞经5-Aza-CdR处理后其RIZ1 mRNA的表达量上调;MTT法检测显示5-Aza-CdR能抑制U87的生长增殖,且与5-Aza-CdR的浓度和作用时间呈负相关。结论：RIZ1基因启动子高甲基化是人脑成胶质细胞瘤细胞株中RIZ1基因表达下调的重要机制。     

CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5' CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.     

Aberrant methylation in promoter-associated CpG islands of multiple genes in relapsed childhood acute lymphoblastic leukemia

Methylation profile was analyzed in nine cases of relapsed childhood acute lymphoblastic leukemia (ALL) for p14, p15, p16, Rb, MGMT, APC, hMLH1, RARbeta, RIZ, DAPK, and FHIT genes by using methylation specific polymerase chain reaction (MSP) analysis. Frequency of methylation in each gene was: MGMT, 56%; RARbeta, 44%; and p16, 22%, respectively. None of the p14, p15, Rb, APC, hMLH1, RIZ, DAPK, and FHIT genes were hypermethylated. Five (56%) of 9 cases showed methylation of at least one gene. All of the samples with hypermethylation in p16 and MGMT gene at relapse, had already acquired the change at the time of initial diagnosis. Interestingly, three of 4 cases with RARbeta gene methylation at relapse did not have methylation of this gene at the time of initial presentation. These results suggest that hypermethylation might be involved in the relapse of childhood ALL.     

Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia

Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.