瘦素启动子甲基化在骨关节炎发病中的作用

目的 探讨瘦素启动子甲基化在骨关节炎(OA)发病中的作用.方法 取OA组和对照组(创伤行截肢手术,除外OA等关节炎性疾病)患者膝骨关节标本,培养膝关节软骨细胞,采用不同浓度(5.0 μmol/L、10.0 μmol/L、20.0 μmol/L)不同时间(12 h、24 h、48 h、72 h、96h、120 h、168 h)5-氮杂-2'-脱氧胞苷(5-Aza-CdR)刺激软骨细胞,实时定量PCR检测膝关节软骨细胞瘦素mRNA表达,同时使用Epityper DNA甲基化分析技术枪测该基因启动子部分区域(-280～+79)的甲基化状态.结果 (1)10μmol/L 5-Aza-CdR刺激OA组软骨细胞,其瘦素mRNA表达水平增高,72 h最为显著.(2)OA组5-Aza-CdR刺激72 h后软骨细胞瘦素mRNA表达最高,对照组无5-Aza-CdR刺激软骨细胞瘦素mRNA表达最低.(3)使用非先导分层聚类分析法对瘦素启动子区域进行分析,2组甲基化模式存在差异,且 5-Aza-CdR刺激前后甲基化模式也不相同.结论 瘦素启动子部分位点的去甲基化可能是引起该基因异常表达导致OA发生的始动因素之一.

Abstract：

Objective To investigate the effects of 5-Aza-CdR( methylation transferase inhibitor) on the expression levels of leptin gene in chondrocytes and methylation states of leptin promoter region between osteoarthritis (OA) group and control. Methods The chondrocytes in osteoarthritis group were treated with 5-Aza-CdR with different doses and time-points, and the expression level of leptin was detected by real-time polymerase chain reaction for picking up the optimum dose and time-point. Next, the chondrocytes in 5 osteoarthritis patients and 5 control patients (amputation due to severe trauma) were treated with 5-Aza-CdR. Lastly, leptin mRNA expression levels in the four groups osteoarthritis and control chondrocytes treated with/without 5-Aza-CdR were measured by real-time PCR and the methylation state of promoter region ( - 280- + 79) was detected by epityper quantitative DNA methylation analysis. Results ( 1 ) After treating the chondrocytes in OA groups with 10 μmol/L 5-Aza-CdR for 72 h, the mRNA expression levels of leptin were increased significantly. ( 2 ) The mRNA expression levels of leptin were significantly different among the four groups ( P ＜ 0. 05 ), and the chondrocytes in osteoarthritis groups treated with 5-Aza-CdR showed a marked induction of leptin mRNA expression. (3) Analysis of quantitative methylation data using an unsupervised hierarchical clustering algorithm, showed that methylation patterns of leptin promoter was different between control and osteoarthritis chondrocyte treated with/without 5-Aza-CdR. Conclusion Demethylation of leptin promoter might up-regulate leptin gene expression level and it might contribute to osteoarthritis.     

Aberrant methylation of death-associated protein kinase 1 CpG islands in myelodysplastic syndromes

Death-associated protein kinase 1 (DAPK1), a proapoptotic serine/threonine kinase, is a candidate tumor suppressor gene. We studied the methylation status of the promoter region of the DAPK1 gene and the expression of the DAPK1 protein in 78 bone marrow samples from untreated patients with myelodysplastic syndrome (MDS) by PCR and Western blot analysis. Hypermethylation of DAPK1 was present in 42.3% (33 of 78) of MDS specimens and was significantly correlated with the loss of DAPK1 mRNA and protein expression (p < 0.01). There were no significant differences in methylation frequency among subgroup of MDS. DAPK1 hypermethylation in MDS was associated with the presence of cytogenetic abnormalities in the bone marrow at the time of the initial diagnosis. A higher frequency of DAPK1 hypermethylation was found in the unfavorable cytogenetic risk group (12 of 21 cases; 57.1%) compared to the favorable cytogenetic risk group (5 of 20 cases; 25.0%, p = 0.0368). These findings suggest that suppression of DAPK1 expression by DNA methylation may play a substantial role in the development of MDS.     

Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs). The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarcinogenesis, and that reduced expression of MBD4 may play a role in malignant progression of HCC.     

胃腺癌凝血栓蛋白1基因异常甲基化及其临床意义

目的　探讨凝血栓蛋白 1(THBS1)基因CpG岛异常甲基化与胃腺癌 (GAC)发生的关联。方法　应用甲基化特异性PCR检测技术 ,检测 82例GAC患者肿瘤组织 ,30例十二指肠球部溃疡 (对照组 )、30例慢性胃炎伴肠上皮化生或异型增生患者胃窦黏膜组织中 ,THBS1基因启动子CpG岛甲基化分布情况。结果　THBS1基因启动子CpG岛甲基化在对照组 (6 .7% )中的频率明显低于胃炎组(2 6 .7% ,χ2 =4 .32 ,P =0 .0 38)和胃癌组 (4 7.6 % ,χ2 =16 .2 ,P <0 .0 0 1) ,在胃炎组与胃癌组之间的频率差异也有显著性 (χ2 =4 .14 ,P =0 .0 4 2 ) ,老年胃癌患者 (6 3.6 % )中的频率明显高于非老年 (36 .7% ,χ2 =5 .72 ,P =0 .0 17) ,在TNMⅢ和Ⅳ期肿瘤 (6 6 .7% )组织中的频率明显高于Ⅰ期 (36 .8% )或Ⅱ期(36 .4 % ,χ2 =6 .93,v =2 ,P =0 .0 31) ,而肿瘤部位之间、Lauren分型肿瘤之间及不同分化程度肿瘤之间 ,THBS1基因甲基化率的差异无显著性。结论　THBS1基因启动子CpG岛甲基化可能与胃腺癌的发生有关 ,且以老年患者及Ⅲ和Ⅳ期肿瘤多见。     

CpG island methylation in gastroenterologic neoplasia: a maturing field

Fifteen years after the first demonstration of epigenetic tumor-suppressor gene inactivation associated with promoter methylation, the field has reached a level of understanding that threatens a re-writing of established biologic concepts. In gastrointestinal malignancies, epigenetic analysis has led to novel hypotheses regarding the etiology of age-associated cancer susceptibility and the interactions between environmental exposures and neoplasia. Methylation profiling has uncovered a distinct pathway to colorectal neoplasia that may arise from a hitherto underestimated precursor lesion, the proximal hyperplastic polyp-serrated adenoma pathway. Epigenetic information has shown promise in clarifying susceptibility to cancer and defining poor prognosis groups in gastrointestinal cancers. Finally, the field has engendered renewed interest in therapeutic targeting of epigenetic regulatory molecules, and several such drugs are currently in clinical trials. It is likely that epigenetic pathways will be integrated in the routine management of gastrointestinal malignancies over the next decade.     

Preferential isolation of DNA fragments associated with CpG islands.

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.     

肝癌细胞p15基因CpG岛甲基化研究

目的通过检测肝癌细胞株HepG2的抑癌基因p15基因启动子区CpG岛的甲基化状态,探讨其与肿瘤发生的可能相关性。方法应用甲基化特异性PCR(MSP)技术,对人肝癌细胞株HepG2的p15基因启动子区域CpG岛的甲基化状态进行检测,以人淋巴瘤细胞株Raji为阳性对照,以正常人外周血单核细胞(PBMC)和肝细胞为阴性对照。结果肝癌细胞HepG2中p15基因启动子区域CpG岛甲基化和非甲基化检测均呈阳性,正常人外周血单核细胞和肝细胞甲基化检测阴性。结论肝癌细胞株HepG2抑癌基因p15基因CpG岛存在高度甲基化,可能与肝癌的发生相关。     

Number of CpG islands and genes in human and mouse.

Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from bulk DNA. Our results suggest that there are approximately 45,000 CpG islands per haploid genome in humans and 37,000 in the mouse. Sequence comparison confirms that about 20% of the human CpG islands are absent from the homologous mouse genes. Analysis of a selection of genes suggests that both human and mouse are losing CpG islands over evolutionary time due to de novo methylation in the germ line followed by CpG loss through mutation. This process appears to be more rapid in rodents. Combining the number of CpG islands with the proportion of island-associated genes, we estimate that the total number of genes per haploid genome is approximately 80,000 in both organisms.     

TIP30基因启动子甲基化与大肠癌细胞5-氟尿嘧啶化疗敏感性的关系

目的探讨TIP30启动子CpG岛甲基化状态与大肠癌细胞对5-氟尿嘧啶(5-Fu)敏感性的关系。方法采用MTT法检测HCT116和HT29大肠癌细胞株对5-Fu的敏感性。应用甲基化特异性PCR(MSP)方法,检测2细胞株对5-Fu敏感性有差异的大肠癌细胞株中TIP30基因启动子CpG岛甲基化状态,并用RT-PCR检测其mRNA的表达水平。结果 TIP30基因在HCT116及HT29癌细胞中甲基化状态有差异,其表达水平与启动子CpG岛甲基化状态有关,并且二者对5-氟尿嘧啶敏感度不同。结论 TIP30基因启动子甲基化状态可能影响大肠癌细胞对化疗药物的敏感性,为大肠癌患者的个体化治疗提供了可能。     

Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation.

We have developed a PCR-based system that allows us to assess the relative frequency of use of specific bases as targets for the avian leukosis virus in vitro integration system. Using this system, we tested the effect of 5-methylation of cytosine in runs of CpG on the distribution of integration target sites. We found that the distribution of preferred integration sites was not uniform along the target DNA; rather, there was a distinct and reproducible pattern of frequently used sites. This pattern was independent of orientation of the integrated DNA, and of overall structure and sequence of the target and fragment amplified. Methylation did not inhibit integration into CpG dinucleotides; on the contrary, this modification created highly preferred targets within runs of alternating CpG. Finally, similar but not identical specificity was observed by using preintegration complexes in infected extracts or purified integrase and DNA as enzyme and substrate. Thus, most of the specificity observed is conferred by interaction of integrase and targets, although it may be modified by other viral and/or cellular components.     

Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay

De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX, LHX, PAX, DLX, and Engrailed, were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7- and HOXA9-associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.     

A novel real-time PCR assay for the speciation of medically important ticks

The identification of ticks using morphological characters is a well-established practice, however specimens that are small or damaged are often difficult to speciate. A novel, rapid real-time PCR assay, which targets the second internal transcribed spacer (ITS2) region in the nuclear ribosomal DNA gene, was developed for identification of four tick species of utmost medical importance in the United States: Ixodes scapularis, I. pacificus, Dermacentor variabilis, and Amblyomma americanum. Computational analyses of public databases and DNA sequencing studies revealed regions that could be specifically targeted with oligonucleotides optimized for TaqMan chemistry. The oligonucleotide sets designed in this study are specific at both the genus and species levels, and are sensitive at 0.1-1 pg of total tick DNA.     

CpG island methylation of tumor-related promoters occurs preferentially in undifferentiated carcinoma.

OBJECTIVE: To understand the role of epigenetic inactivation of tumor-related genes in the pathogenesis of thyroid cancer, we investigated the methylation profile of distinct thyroid neoplasms. DESIGN: We analyzed the methylation pattern of 17 gene promoters in nine thyroid cancer cell lines and in 38 primary thyroid carcinomas (13 papillary thyroid carcinoma [PTC], 10 follicular thyroid carcinoma [FTC], 9 undifferentiated thyroid carcinoma [UTC], 6 medullary thyroid carcinoma [MTC]), 12 goiters, and 10 follicular adenomas (FA) by methylation- specific polymerase chain reaction (PCR). Epigenetic inactivation was validated by expression analysis. MAIN OUTCOME: Twelve of these genes (RASSF1A, p16(INK4A), TSHR, MGMT, DAPK, ERalpha, ERbeta, RARbeta, PTEN, CD26, SLC5A8, and UCHL1) were frequently methylated in UTC (15%-86%) and thyroid cancer cell lines (25%-100%). In the more aggressive UTC, the mean methylation index (MI = 0.44) was the highest compared to other thyroid alterations PTC (MI = 0.29, p = 0.123), FTC (MI = 0.15, p = 0.005), MTC (MI = 0.13; p = 0.017), FA (MI = 0.27; p = 0.075) and goiters (MI = 0.23; p = 0.024). Methylation of TSHR, MGMT, UCHL1, and p16 occurred preferentially in UTC and this inactivation was reverted by a demethylating agent. CONCLUSIONS: Our results show that hypermethylation of several tumor-related gene promoters is a frequent event in UTC. The hypermethylation status may be reversed by DNA demethylating agents. Their clinical value remains to be investigated.     

Analysis of promoter methylation in stool: a novel method for the detection of colorectal cancer.

BACKGROUND & AIMS: Detection of tumor-derived DNA alterations in stool is an intriguing new approach with high potential for the noninvasive detection of colorectal cancer (CRC). Because of heterogeneity of tumors, usually multiple markers distributed throughout the human genome need to be analyzed. This is labor intensive and does not allow for high through-put screening. Therefore, markers with high sensitivity and good specificity are needed. We explored the potential of a single epigenetic marker in comparison with fecal occult blood testing (FOBT) for the discrimination of patients with CRCs and adenomas from those without. METHODS: Methylation-specific polymerase chain reaction (PCR) was performed to analyze hypermethylated in cancer 1 (HIC1) promoter methylation status in a blinded fashion in stool samples from 26 patients with CRC, 13 with adenoma > or =1 cm, 9 with hyperplastic polyps, 9 with chronic inflammatory bowel disease, and 32 with endoscopically normal colon. RESULTS: Ninety-seven percent of the stool samples contained amplifiable DNA. Forty-two percent of the samples from patients with CRC and 31% of the samples from patients with colorectal adenoma > or =1 cm were positive for HIC1 promoter methylation. No methylated HIC1 promoter DNA was detected in the fecal DNA from patients with endoscopically normal colon or hyperplastic polyps. CONCLUSIONS: The epigenetic marker HIC1 promoter methylation carries high potential for the remote detection of CRCs. We postulate that a panel of merely a few genetic and epigenetic markers will be required for the highly sensitive and specific detection of CRCs and adenomas in fecal samples from affected patients.     

实时荧光定量PCR和甲基化特异性PCR检测结直肠癌APC、MLH1基因甲基化状态

背景：启动子区甲基化致肿瘤抑制基因失活在结直肠癌的发生、发展中起重要作用,检测肿瘤相关基因的甲基化状态,可能为寻找新的结直肠癌诊断、预后相关标记物提供依据.目的：比较实时荧光定量PCR（FO-PCR）与甲基化特异性PCR（MSP）检测基因甲基化状态的差异.方法：以FQ-PCR检测66例结直肠癌组织和20例癌旁组织中与结直肠癌的发生、发展相关的抑癌基因APC和错配修复基因MLH1的甲基化状态,同时以MSP检测结直肠癌组织中APC基因的甲基化状态.结果：根据FQ-PCR结果,结直肠癌组织中APC、MLH1基因甲基化阳性率显著高于癌旁组织（48.5%和54.5%对0%和0%,P=0.000）.FQ-PCR和MSP可检出的甲基化阳性对照DNA最低浓度分别为0.015 ng/μl和1.5 ng/μl,两者对APC基因甲基化状态的检测结果差异有统计学意义（P＜0.05）.结论：在DNA甲基化的检测手段中,FQ-PCR的敏感度优于MSP.