食管癌组织中金属硫蛋白3基因甲基化的地区性差异

目的:探讨食管癌高发区河北省中南部及广东省潮汕地区和食管癌非高发区的食管癌及正常组织中金属硫蛋白3 (metallothionein-3, MT-3)基因CpG岛的甲基化情况及其临床意义.方法:选取正常人血液标本10例,胎儿食管组织标本10例,非食管癌高发区正常人食管黏膜标本20例,河北省中南部食管癌高发区和潮汕地区食管癌新鲜手术标本及切缘正常组织各30例;应用甲基化特异性聚合酶链反应 (methylation-specific PCR, MSP)检测其MT-3基因CpG岛的甲基化情况,并分析其与临床病理资料之间的关系.结果: 高发区食管癌患者的切缘正常组织有20例(33.3%)存在MT-3基因甲基化,高于非高发区正常人食管黏膜(P=0.013);食管癌组织中有49例(81.7%)甲基化,明显高于相应肿瘤切缘正常组织(P<0.001),但2个高发区之间比较差异无统计学意义(P=0.739).结论:食管癌患者的正常食管黏膜及癌组织中均广泛存在MT-3基因的甲基化,后天因素刺激可能是导致MT-3基因甲基化的重要原因.     

新疆哈萨克族食管癌survivin启动子区CpG岛甲基化状态及其临床意义

目的： 检测消化道肿瘤早期相关基因survivin mRNA在哈萨克族食管癌组织中的表达及其启动子区CpG岛甲基化状态,并进一步探究survivin启动子甲基化是否参与了食管癌的发生。方法：提取哈萨克族食管癌患者癌组织及远端无癌组织RNA,RT-PCR检测组织中survivin mRNA的表达水平,甲基化特异性PCR (methylation specific PCR,MSP)检测survivin启动子区CpG 岛的甲基化状态。结果：在20对食管癌组织标本中,癌组织中survivin mRNA阳性表达率 (95%)明显高于远端无癌组织 (65%) (P＜0.05)。癌组织及远端无癌组织中survivin基因启动子区CpG岛多为杂合型甲基化,且两种组织间甲基化状态无明显差异 (P＞0.05)。结论：survivin mRNA表达水平的增高在哈萨克族食管癌的发生、发展过程中起到了一定的作用,但其表达与启动子区CpG岛甲基化无关,提示甲基化并未直接调控该基因的表达并促进哈萨克族食管癌的发生发展。     

CHFR基因CpG岛甲基化对食管癌Eca109细胞增殖和凋亡的影响

目的:研究CHFR基因启动子甲基化状态与食管癌Eca109细胞增殖和凋亡的关系。方法:分别用不同浓度(2、5、10μmol/L)5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)处理Eca109细胞,未经药物处理的细胞为对照组。甲基化特异性PCR(methylation-specific PCR,MSP)检测CHRF基因CpG岛的甲基化状态,RT-PCR检测CHFR mRNA的表达情况,MTT法及流式细胞术检测不同浓度5-Aza-CdR处理对Eca109细胞增殖及凋亡的影响。结果:对照组Eca109细胞CHFR CpG岛处于高甲基化状态,5-Aza-CdR处理后CHFR CpG岛可发生不同程度的剂量依赖性的去甲基化。对照组Eca109细胞无CHFR mRNA表达,5-Aza-CdR处理组(2、5、10μmol/L)CHFR mRNA相对表达量显著增加(0.174±0.010、0.221±0.013、0.356±0.014)。不同浓度5-Aza-CdR处理后,Eca109细胞增殖受到抑制[作用72 h时的抑制率分别为(30.87±0.74)%、(44.60±0.79)%、(56.67±0.35)%],细胞凋亡显著增加[(7.46±1.46)%、(16.27±1.61)%、(25.29±2.25)%vs(1.83±0.41)%,P<0.01]。结论:食管癌Eca109细胞经5-Aza-CdR处理后,CHFR基因CpG岛发生部分去甲基化,出现CHFRmRNA表达,并抑制Eca109细胞增殖和促进细胞凋亡。     

新疆哈萨克族和汉族食管癌患者金属硫蛋白-3 CpG岛甲基化的研究

目的： 检测分析和比较新疆哈萨克族和汉族食管癌及癌旁组织中金属硫蛋白-3 (metallothionein-3,MT-3)基因启动区CpG岛的甲基化情况,为寻找新疆哈萨克族食管癌诊断治疗的分子标记物提供线索。方法：采用甲基化特异性聚合酶链反应 (methylation specific polymerase chain reaction,MSP)的方法,检测33例哈萨克族和30例汉族食管癌患者食管癌组织和癌旁组织中MT-3 基因启动子区CpG岛的甲基化发生状况。 结果：33例哈萨克族食管癌组织和癌旁组织标本中MT-3基因启动子区甲基化发生率分别为66.7% (22/33)和27.3% (9/33),差异有统计学意义 (χ2=10.280,P=0.001);30例汉族食管癌组织和癌旁组织标本中MT-3基因启动子区甲基化发生率分别为56.7% (17/30)和26.7% (8/30),差异亦有统计学意义 (χ2=5.554,P=0.018)。哈、汉民族之间癌组织和癌旁组织MT-3基因启动子区甲基化率的差异均无统计学意义 (χ2=0.666,P=0. 414;χ2=0.003,P=0.957)。 结论：MT-3基因启动子区CpG岛的甲基化可能与食管癌的发生与发展有关,但不是哈族食管癌患者的特异性致病因素。     

肝细胞癌中ChREBP基因CpG岛甲基化与mRNA表达的相关性

目的 检测肝细胞癌中ChREBP基因CpG岛甲基化水平及mRNA表达水平,并探讨两者的相关性。方法 收集肝细胞癌及癌旁冰冻组织90例,采用亚硫酸氢钠处理结合克隆测序检测ChREBP基因CpG岛内29个CpG位点的甲基化水平;采用荧光定量PCR检测其中31对新鲜冰冻组织中ChREBP基因mRNA表达水平,分析甲基化水平与mRNA表达之间的关系。结果 ChREBP基因的5、6、7、14号CpG位点甲基化水平在肝细胞癌中显著低于其配对的癌旁组织（P<0.05）。其余CpG位点及整体甲基化在肝细胞癌与癌旁组织中差异无统计学意义（均P>0.05）。15、18、20、23、26、29号CpG位点在≥50岁年龄组的甲基化水平均高于<50岁年龄组（均P<0.05）。肝细胞癌中ChREBP基因mRNA表达水平低于癌旁组织（P=0.003）,但与甲基化无显著相关性（P>0.05）。结论 肝细胞癌中ChREBP基因mRNA表达水平低于癌旁组织,但这一改变可能不是通过影响DNA的甲基化水平实现的。     

DNA修复酶基因MGMT启动子区异常甲基化与食管癌的关系

背景与目的： 分析食管癌组织中MGMT启动子区CpG岛甲基化状态和食管癌发病风险的关系,探讨MGMT基因启动子的异常甲基化在食管癌筛查及早期诊断中的意义。 材料与方法： 对江苏淮安的91例新发食管癌病例的癌组织、癌旁组织及外周血血浆样本提取DNA,应用甲基化特异性PCR(MSP)分析MGMT启动子区CpG岛的甲基化状态;对食管癌组织和癌旁组织提取总RNA,采用SYBR GREEN I 实时荧光定量逆转录-聚合酶链反应(RT-PCR)测定MGMT的mRNA水平。 结果： MGMT启动子区CpG岛异常甲基化与食管癌发病风险增高有关联 (OR=7.750, 95％CI=2.736～21.955);MGMT启动子区CpG岛甲基化状态与MGMT mRNA水平无显著相关;血浆循环DNA中MGMT启动子区CpG岛甲基化与癌组织中MGMT启动子区CpG岛甲基化相关(P<0.01),血浆循环DNA中MGMT启动子区CpG岛甲基化检出率与癌组织中MGMT启动子区CpG岛甲基化检出率中度相关(Kappa=0.603, P<0.01)。 结论： MGMT基因的启动子区CpG岛的异常甲基化与食管癌发病风险增加有关;检测血浆循环DNA中MGMT基因启动子的异常甲基化,可为食管癌的筛查、早期诊断提供有价值的信息。     

细胞信号转导抑制因子1基因超甲基化及去甲基化在经典型骨髓增殖性肿瘤中的作用

目的:探讨细胞信号转导抑制因子1(suppressor of cytokine signaling1,SOCS1)基因超甲基化在经典型慢性骨髓增殖性肿瘤(myelo-proliferative neoplasms,MPNs)中的临床作用及其机制。方法:采用甲基化特异性PCR法检测MPNs患者骨髓标本中SOCS1基因的超甲基化发生情况。应用粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)化学诱导MPNs细胞生长不同时间后,实时定量PCR和蛋白质印迹法检测SOCS1mRNA及其蛋白表达情况。另外,加入去甲基化试剂2-deoxyazacytidin培养MPNs细胞不同时间后,实时定量PCR法检测SOCS1mRNA表达情况。结果:100例经典型慢性MPNs患者中有27例(27.0%)存在SOCS1基因超甲基化。G-CSF化学诱导处理后的MPNs细胞中,SOCS1基因超甲基化组与未甲基化组相比,其SOCS1mRNA表达量均明显减少(均P<0.05);MPNs患者中SOCS1基因超甲基化组的SOCS1蛋白表达量也减少,但与健康对照组相比差异尚无统计学意义(P>0.05)。另外,相对于G-CSF处理后的超甲基化组,加入去甲基化试剂后SOCS1mRNA表达量明显升高(P<0.05)。结论:MPNs患者中存在SOCS1基因超甲基化,且CpG岛超甲基化导致其基因表达水平降低,去甲基化后,其基因表达水平升高。提示SOCS1基因超甲基化可能和MPNs发病有关,去甲基化可能是一种潜在的治疗MPNs的新策略。     

抗凋亡基因bcl-2启动子CpG岛的高甲基化状态见于28.3%(13/46例)的膀胱癌患者尿沉淀细胞DNA

目的:检测上海地区膀胱癌患者尿沉淀细胞中3个肿瘤相关基因启动子CpG岛的甲基化谱式异常频率,继而评估其应用前景。方法:采用甲基化特异性PCR方法分析尿沉淀细胞DNA中DAPK1、bcl2和hTERT3个基因的启动子CpG岛甲基化状态,并通过RTPCR方法评估DAPK1基因在膀胱癌细胞系中的表达状态。结果:对膀胱癌细胞系中DAPK1基因的启动子CpG岛甲基化状态及其表达(mRNA水平上)所做的分析,确立了高甲基化状态与表达静息化之间的相关性。对46例临床确诊的膀胱癌患者和84例非膀胱癌对照(包括前46例术后的36例)的尿沉淀细胞中DNA甲基化的分析发现,仅bcl2基因的高甲基化见之于28.3%(13/46例)的膀胱癌患者,而84例的对照中均为去甲基化状态。结论:在美国膀胱癌患者尿沉淀细胞中频发DNA高甲基化的靶点在上海地区膀胱癌患者人群中频率很低,因此寻找在后者中频发DNA高甲基化的新靶点实属必要。     

长链非编码RNA XLOC_005009在食管鳞状细胞癌中的表达及其甲基化状态

[目的]通过检测长链非编码RNA XLOC 005009在人食管癌细胞系及食管鳞状细胞癌(ESCC)组织中的表达情况及其甲基化状态,探讨XLOC_005009基因在食管鳞癌发生、发展中的作用及表观遗传学失活机制.[方法]分别应用逆转录一聚合酶链反应以及甲基化特异性聚合酶链反应的方法检测XLOC_005009基因在DNA甲基化转移酶抑制剂5-Aza-dC处理前后的食管癌细胞系(TE1、TE13、T.Tn、Eca109)以及57例食管鳞癌组织及相应癌旁正常组织中的表达情况和各位点的甲基化状态.[结果] XLOC_005009基因的表达在5-Aza-dC处理后的4株细胞系中表达增高,在5-Aza-dC处理前的4株细胞系中XLOC_005009基因的3个位点均表现为高甲基化状态,在5-Aza-dC处理后,各位点甲基化程度均降低.XLOC 005009基因在ESCC中的表达明显低于对应癌旁正常组织(0.21±0.22 vs 0.32±0.29,P＜0.05),其远端CpG岛及第2外显子区甲基化率在ESCC与其癌旁正常组织中差异无统计学意义,且其在发生甲基化的ESCC与未发生甲基化的ESCC中的表达无统计学差异.第一外显子区甲基化率在ESCC中显著高于其癌旁正常组织[45.61％(26/57)vs 19.30％(11/57),P＜0.05],并与淋巴结转移、组织分化程度及TNM分期密切相关,且此位点发生甲基化的ESCC中该基因的表达显著低于未发生甲基化的ESCC组织(0.06±0.06 vs 0.33±0.23,P＜0.05).[结论]XLOC_005009基因在食管癌细胞系和ESCC中的低表达与ESCC的发生、发展密切相关,且其第一外显子区甲基化异常增高可能是导致其表达沉默的机制之一.     

三氧化二砷对乳腺癌细胞MDA-MB-231雌激素受体α的去甲基化作用

目的研究三氧化二砷（As2O3）作用于乳腺癌细胞MDA-MB-231后,对雌激素受体α（ERα）表达的影响,并初步探讨其机制。方法用As2O3处理ERα阴性的人乳腺癌细胞MDA-MB-231,甲基化特异性PCR（MSP）检测ERα启动子CpG岛的甲基化状态,反转录PCR（RT-PCR）检测ERα mRNA表达水平的变化。以雌激素受体α阳性的人乳腺癌细胞MCF-7 为阳性对照。结果MDA-MB-231细胞ERα启动子CpG岛存在高甲基化,经过As2O3处理后,CpG岛高甲基化水平降低,ERα mRNA恢复表达。结论 一定浓度的As2O3能够使人乳腺癌细胞MDA-MB-231 ERα启动子中的CpG岛发生去甲基化,重新恢复mRNA的表达。     

Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

Hypermethylation of the spleen tyrosine kinase promoter in T-lineage acute lymphoblastic leukemia

Sequence analysis of the noncoding first exon (exon 1) of the Syk gene demonstrated the presence of a previously cloned CpG island (GenBank #Z 65706). Transient transfection analysis in Daudi cells demonstrated promoter activity (18-fold increase over parental luciferase plasmid) for a 348 bp BstXI-BsrBI fragment containing this island. This region exhibits a high GC content (approximately 75%), contains several SP1 binding sites and a potential initiator sequence, but lacks a strong TATA consensus. Bisulfite sequencing and methylation-specific PCR (MSP) of this region demonstrated that the Syk promoter CpG island was largely unmethylated in B-lineage leukemia cell lines, control peripheral blood cells, human thymocytes and CD3(+) T lymphocytes. However, dense methylation was seen in four T-lineage leukemia cell lines, Jurkat, H9, Molt 3 and HUT 78. MSP screening of leukemia cells from six T-lineage acute lymphoblastic leukemia (ALL) patients demonstrated methylation of the Syk promoter CpG island in one T-lineage ALL patient. Promoter methylation was correlated with reduced to absent expression of Syk mRNA and SYK protein in the T-lineage leukemia cell lines. Treatment of the leukemia lines Ha and Molt 3, with the methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-CdR) resulted in increased Syk mRNA expression. The presence of a methylated promoter sequence in these T-lineage leukemia cell lines and in one T-lineage patient suggests a potential role for SYK as a tumor suppressor in T-ALL.     

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.     

Silencing of bidirectional promoters by DNA methylation in tumorigenesis

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.     

Downregulation of human kallikrein 10 (KLK10/NES1) by CpG island hypermethylation in breast, ovarian and prostate cancers.

OBJECTIVE: The human kallikrein 10 (KLK10)/normal epithelial cell-specific-1 (NES1) gene is highly expressed in normal mammary, ovary and prostate cells, but its expression is dramatically decreased in cancer cell lines. Recently, it has been shown that CpG island hypermethylation of the KLK10 gene is responsible for the tumor-specific loss of KLK10 gene expression in certain breast cancer cell lines. METHOD: We examined the role of CpG island hypermethylation in the tumor-specific loss of KLK10 expression in breast, ovarian and prostate cancers. We treated cells with the demethylating agent 5-aza-2'-deoxycytidine (dC) and monitored changes in KLK10 mRNA by RT-PCR and secreted hK10 protein expression by ELISA. The following cell lines were used: MDA-MB-231, MDA-MB-468, MCF-7, ZR-75-1, T-47D and BT-474 (breast); BG-1, MDAH-2774, HTB-75, HTB-161, PA-1 and ES-2 (ovary), and LNCaP and PC-3 (prostate). RESULTS: Upregulation of KLK10 mRNA levels, which was accompanied by an increase in secreted hK10 protein concentration, was observed for a subset of breast, ovarian, and prostate tumor cell lines after 5-aza-2'-dC. Genomic sequencing of sodium-bisulfite-treated DNA demonstrated that CpG sites within the KLK10 gene exon 3 were highly methylated. Hypermethylation of exon 3 CpG regions was also detected in primary ovarian cancers. CONCLUSION: These data suggest that CpG island hypermethylation plays an important role in the downregulation of kallikrein 10 mRNA and protein expression, but it cannot explain the pattern of expression of this gene in all cell lines or tissue tested.