TIMP-3甲基化调控与大肠癌转移的关系研究

目的探讨TIMP-3基因启动子5’CpG岛甲基化状况与大肠癌转移的关系。方法随机收集45例大肠癌病例。用甲基化特异性PCR检测TIMP-3基因启动子5’CpG岛甲基化状况;用免疫组化方法检测TIMP-3蛋白的表达水平;分析TIMP-3基因启动子甲基化与大肠癌转移之间的关系。结果大肠癌肝转移组、淋巴结转移组TIMP-3基因甲基化率分别高于非肝转移组（P〈0．01）、非淋巴结转移组（P〈0．01）;Duck＇s C＋D组甲基化率高于Duck’s A＋B组。大肠癌TIMP-3基因甲基化与TIMP-3蛋白的表达缺失有关（P〈0．01）。结论TIMP-3甲基化是大肠癌中TIMP-3蛋白表达缺失的主要原因,在大肠癌转移中发挥重要的作用,提示预后不良。     

Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene

Over 50% of human genes are associated with CpG islands and DNA methylation within such CpG islands has been clearly correlated with inhibition of expression. Whereas changes in DNA methylation play a key role in a number of human diseases, in particular cancer, in normal DNA CpG islands are nearly always methylation free, regardless of the expression status of the associated gene. Only limited evidence supports a role for DNA methylation in controlling tissue-specific expression in adult somatic tissue. Loss of expression of the MCJ gene has previously been linked to increased chemotherapeutic drug resistance in ovarian cancer. We report that loss of expression of MCJ in drug-resistant ovarian cancer cell lines depends on methylation of a CpG island within its first exon, but is independent of methylation within the promoter region. Furthermore, cell type-specific expression of the MCJ gene in normal cells also depends on the methylation status of the CpG island within its first exon. The MCJ CpG island is methylated and the gene is not expressed in cells of epithelial origin, but unmethylated and expressed in cells of lymphocyte or fibroblast origin. Chromatin immunoprecipitation assays determined that MCJ CpG island methylation was associated with loss of histone acetylation in ovarian epithelial cells compared with unmethylated fibroblast cells. Reduced acetylation was observed not only within the CpG island, but also within the promoter region, suggesting that CpG island methylation may direct alterations in chromatin structure within the promoter region, leading to gene inactivation.     

TIMP-3基因甲基化调控与大肠癌发生发展的关系研究

目的：探讨TIMP-3甲基化调控与大肠癌发生发展的关系。方法：随机收集45例大肠癌病例、42例大肠腺瘤病例、48例非肿瘤性大肠病例标本,用甲基化特异性PCR（methylation—specific PCR,MSP）分别检测TIMP-3基因启动子5＇CpG岛甲基化状况。结果：非肿瘤性大肠病变、大肠腺瘤、大肠癌分别有0例（0．00％）、5例（11．90％）、14例（31．11％）TIMP-3基因启动子发生甲基化。大肠癌组织TIMP-3甲基化率高于非肿瘤性大肠病变（P〈0．01）和大肠腺瘤（P〈0．05）。大肠腺瘤TIMP-3甲基化率高于非肿瘤性大肠病变（P〈0．05）。大肠癌肝转移组、淋巴结转移组TIMP-3基因甲基化率分别高于非肝转移组（P〈0．01）、非淋巴结转移组（P〈0．01）;Duck＇s C＋D组甲基化率分别高于DuckhA＋B组（P〈0．01）。结论：大肠癌TIMP-3基因的高甲基化在大肠癌的发生、发展中发挥重要作用,对于大肠癌的早期预防、诊断、恶性程度及预后判断有重要意义。     

TIMP-3基因甲基化调控与大肠癌发生发展的关系研究

目的:探讨TIMP-3甲基化调控与大肠癌发生发展的关系.方法:随机收集45例大肠癌病例、42例大肠腺瘤病例、48例非肿瘤性大肠病例标本,用甲基化特异性PCR(methylation-specific PCR,MSP)分别检测TIMP-3基因启动子5'CpG岛甲基化状况.结果:非肿瘤性大肠病变、大肠腺瘤、大肠癌分别有0例 (0.00%)、5例 (11.90%)、14例(31.11%)TIMP-3基因启动子发生甲基化.大肠癌组织TIMP-3甲基化率高于非肿瘤性大肠病变(P＜ 0.01)和大肠腺瘤(P＜ 0.05).大肠腺瘤TIMP-3甲基化率高于非肿瘤性大肠病变(P＜ 0.05).大肠癌肝转移组、淋巴结转移组TIMP-3基因甲基化率分别高于非肝转移组(P＜ 0.01)、非淋巴结转移组(P＜ 0.01);Duck's C+D组甲基化率分别高于Duck's A+B组(P＜ 0.01).结论:大肠癌TIMP-3基因的高甲基化在大肠癌的发生、发展中发挥重要作用,对于大肠癌的早期预防、诊断、恶性程度及预后判断有重要意义.     

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.     

上皮性卵巢癌组织中WWOX基因启动子区域CpG岛的甲基化状态及临床意义

目的研究上皮性卵巢癌组织中WWOX基因启动子区CpG岛的甲基化状态,并分析WWOX基因的甲基化与上皮性卵巢癌的临床病理指标之间的关系。方法采用甲基化特异性PCR(methylation specific polymerase chain reaction,MSP)方法检测48例上皮性卵巢癌、18例卵巢交界性上皮性肿瘤、26例卵巢良性上皮性肿瘤及33例正常卵巢组织中WWOX基因CpG岛甲基化状态。结果上皮性卵巢癌、卵巢交界性上皮性肿瘤、卵巢良性上皮性肿瘤组织中WWOX基因启动子区CpG岛甲基化率分别为43.75%、26.32%、3.84%,正常卵巢组织中未检测到WWOX基因CpG岛甲基化。上皮性卵巢癌组织中WWOX基因CpG岛的甲基化率明显高于其他卵巢组织,差异有统计学意义(P<0.01)。晚期(Ⅲ期、Ⅳ期)上皮性卵巢癌组织中WWOX基因CpG岛的甲基化率高于早期(Ⅰ期、Ⅱ期)上皮性卵巢癌组织,差异有统计学意义(P<0.05)。结论 上皮性卵巢癌组织中广泛存在着WWOX基因启动子区CpG岛甲基化,可能是导致WWOX基因失活的重要机制。WWOX基因的异常甲基化可能与上皮性卵巢癌的发生发展密切相关,其可能成为上皮性卵巢癌的早期诊断和评估预后的重要指标。     

3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性

目的对3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性进行评估.方法通过甲基化特异性多聚酶链反应法(MSP)结合DNA测序来确定3株肺腺癌细胞株(A549,SH77和SPE-A-1)中9个基因的启动子CpG岛的甲基化状态:APC,CDH13,DAPK1,MGMT,MYOD1,p16INK4a,p73,RAR-β和WT1基因.同时采用半定量RT-PCR的方法来量度下列5个基因的表达:CDH13,MGMT,MYOD1,RAR-β和WT1基因.结果在9个受检基因之中,下列4个基因在三株细胞中均呈同样的甲基化谱式:仅有去甲基化:APC和DAPK1,以及兼有甲基化和去甲基化:p73和p16INK4a.而其余5个基因则表现为不同的甲基化模式.尽管,这5个基因的表达谱式大体上符合其启动子CpG岛甲基化的表达静息的原则,个别的例外亦是有的.这提示着与DNA甲基化状态无关的机制可能也会参与该基因的表达调控.结论肺癌细胞株中的肿瘤相关基因的启动子CpG岛的过甲基化可与该基因的表达状态有较高的相关性.     

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.     

GSTP1基因在前列腺癌组织中的表达及甲基化状态研究

目的：研究谷胱苷肽S转移酶P1（glutathione S transferase pi,GSTP1）在前列腺癌的表达情况并分析其甲基化状态.方法：采用免疫组织化学方法检测GSTP1在50例前列腺癌石蜡标本中的表达情况,并用甲基化特异性聚合酶链反应（methylation-specific polymerase chain reaction, MSP）方法分析GSTP1基因启动子区5＇端CpG岛非甲基化和甲基化的状态.结果： GSTP1在前列腺癌表达的阳性率为18.0%,明显低于癌旁正常前列腺腺体的阳性率（76.0%）,P=0.000.GSTP1基因启动子区非甲基化发生率为86%（43/50）,50例前列腺癌均未扩增出甲基化特异性产物.结论： GSTP1阴性表达有望成为前列腺癌诊断的一项辅助性新指标.国内前列腺癌GSTP1基因的高频率表达缺失可能与GSTP1基因启动子区甲基化无关,因本组病例数较少,还需进一步积累资料证实.     

长链非编码RNA ZNF667-AS1在食管鳞状细胞癌组织中的表达及其DNA甲基化状态的研究

 目的 检测长链非编码RNA ZNF667-AS1在食管鳞状细胞癌（esophageal squamous cell carcinoma, ESCC）中的表达及甲基化状态。方法 应用qPCR和MSP法检测DNA甲基化转移酶抑制剂5-Aza-dC处理前后食管癌细胞系（Kyse170、Eca109、TE1和TE13）、ESCC及相应癌旁正常组织中ZNF667-AS1基因的表达,及其CpG岛三个区域的甲基化状态。结果 在5-Aza-dC处理后的四株细胞系中,ZNF667-AS1基因的表达均增高,且其CpG岛三个区域甲基化程度均降低。ZNF667-AS1基因在ESCC组织中的表达显著低于相应癌旁正常组织,且该基因CpG岛远端启动子区及第一外显子区甲基化率在ESCC与相应癌旁正常组织中的差异均具有统计学意义（均P<0.05）。ESCC组织中CpG岛近端启动子区甲基化率显著高于相应癌旁正常组织,并与组织分化程度、TNM分期密切相关,ZNF667-AS1基因CpG岛近端启动子区发生甲基化的ESCC组织中,低表达例数高于高表达例数,差异具有统计学意义。结论 ZNF667-AS1基因在食管癌细胞系和ESCC组织中的低表达与ESCC的发生发展密切相关,且其近端启动子区的高甲基化状态可能是导 致其表达沉默的机制之一。