Methylation status of the PPP1R13L promoter region among lung cancer patients and healthy controls. Analytical cross-sectional study

ABSTRACT BACKGROUND: There is evidence that genetic predisposition and epigenetic alteration (e.g. DNA methylation) play major roles in lung cancer. In our genetic epidemiological studies, rs1970764 in oncogene PPP1R13L was most consistently associated with lung cancer risk. Here, we explored the role of PPP1R13L methylation in lung cancer development. DESIGN AND SETTING: Analytical cross-sectional study (45 lung cancer cases and 45 controls), conducted in China. METHODS: We investigated the DNA methylation status of 2,160 cytosine-phosphate-guanine (CpG) sites in the PPP1R13L promoter region using the EpiTYPER assay of the Sequenom MassARRAY platform. RESULTS: In the whole study group, the methylation levels of CpG-6, CpG-9, CpG-20 and CpG-21 were significantly lower and those of CpG-16 were significantly higher in cases than in controls. Among smokers, the methylation levels at five CpG sites (CpG-6, CpG-11, CpG-15, CpG-20 and CpG-21) were statistically significantly lower among cases. Among men, the methylation levels at four CpG sites (CpG-11, CpG-15, CpG-20 and CpG-21) were significantly lower among cases. Regarding smokers, the methylation levels at CpG-7.8 and CpG-21 among cases and at CpG-22 among controls were significantly lower, compared with nonsmokers. The frequency of positivity for methylation was not significantly different between lung cancer cases and controls (68.22% for cases and 71.87% for controls; P = 0.119). CONCLUSION: Our study on a Chinese population suggests that lung cancer patients have aberrant methylation status (hypomethylation tended to be more frequent) in peripheral blood leukocytes at several CpG sites in the PPP1R13L promoter region and that exposure to smoking may influence methylation status.

Relationship between DNA methylation and digestive system malignant tumors / 肿瘤研究与临床

DNA methylation refers to the use of S-adenosylmethionine as a methyl donor(SAM),in the role of DNA methyltransferases(DNMT),the methyl group added 5-methylcytosine(5-mC)was formed on the cytosine of 5'terminal CpG dinucleotide.DNA methylation is one of the common epigenetic events,play a key role in the diagnosis and treatment of tumors,increasingly become a hot topic of related tumors.Digestive system tumors is the main cause of cancer-related death worldwide,and there is a lack of early diagnosis.This article provides a brief overview of the relationship between DNA methylation and digestive system malignant tumors.

Experimental research on methylation status of CpG islands in promoter region of TGF-β3 and Dnmts during TCDD-induced fetal palatogenisis / 中华整形外科杂志

Objective@#To investigate the correlation between CpG islands methylation statuses of TGF-β3, Dnmts and their expression during TCDD-induced mouse embryonic palatal development.@*Methods@#Eithtteen pregnant C57BL/6J mice were randomly divided into 2 groups: the control group(n=9) and TCDD-exposure group(n=9). At gestation day 10.5(GD10.5), the mice in TCDD-group were orally administrated with TCDD 28 μg/kg, while the mice in the control group received equivalent corn oil. The pregnant mice were sacrificed at GD13.5, GD14.5, GD15.5, fetal palates were collected. CpG island methylation statuses were analysed by methylation specific polymerase chain reaction(MSP). IBM SPSS 20.0 software was applied for statistical analysis. Kolmogorov-Smirnov test was used for normal distribution check, and the distributions were normal. Independent t-test was carried out between two groups. P<0.05 was considered statistically significant.@*Results@#CpG island in promoter region of gene TGF-β3 were all at low methylation level at all GDs of both groups, there were no differences at same GD between two groups [GD13.5: (8.6±0.8)% vs (8.7±0.8)%, P>0.05; GD14.5: (11.5±1.4)%vs (11.7±1.0)%, P>0.05; GD15.5: (12.0±0.7)% vs (12.1±0.5)%, P>0.05]. CpG island in promoter region of gene Dnmt1 were all highly methylated with no differences showed at same GD between two groups [GD13.5: (73.9±1.1)%vs (72.6±0.8)%, P>0.05; GD14.5: (70.8±1.7)% vs (70.7±1.0)%, P>0.05; GD15.5: (69.4±2.2)% vs (69.7±0.5)%, P>0.05]. The methylation level of CpG island 1 in promoter region of gene Dnmt3a in TCDD group was higher than that in control group at GD13.5 and GD15.5 [(21.9±1.1)% vs (8.1±0.6)%, P<0.01, (43.4±0.4)% vs(32.9±0.7)%, P<0.01], while lower at GD14.5[(33.2±0.5)% vs (42.9±0.3)%, P<0.01]. The methylation level of CpG island 2 in promoter region of gene Dnmt3a in control group was higher than that in TCDD group at all GDs [GD13.5: (82.0±0.7)% vs (32.3±0.6)%, P<0.01; GD14.5: (62.7±1.0)%vs (25.5±1.4)%, P<0.01; GD15.5: (47.2±0.4)% vs (30.3±1.4)%, P<0.01].@*Conclusions@#Low methylation level of CpG island 2 which is close to the first exon in promoter region of gene Dnm3a may be the cause of highly expressed Dnmt3a mRNA at GD13.5 during mice palatogenesis induced by TCDD, thus the global DNA methylation is extremely high to induce cleft palate. TCDD-treatment doesn′t influence the CpG methylation statuses in promoter region of TGF-β3 and Dnmt1.

Prediction of CpG islands and functional verification on promoters of FPS, SS, SE in Eleutherococcus senticosus / 中草药

Objective: To obtain the distribution and functional activity of CpG islands in promoters of FPS, SS, and SE from Eleutherococcus Senticosus. Methods: Based on the promoter sequence of FPS, SS and SE, CpG islands were predicted by using EMBOSS and Li Lab. The functional verification of transformed Arabidopsis thaliana was mediated by Agrobacterium tumefaciens by using GUS and pCAMBIA1301 plasmid as the reporter gene and expression vector respectively. Results: Two CpG islands were found in FPS and SS promoter with the lengths of 520 bp, 218 bp and 108 bp, 103 bp, and three CpG islands in SE promoter in 290 bp, 119 bp and 149bp. The promoters of FPS, SS and SE all had promoter activities at different level, in which SS promoter was the highest one. Conclusion: The functional verification and distributions of CpG islands in the promoters area of FPS, SS, and SE were reported in this research at first time, which established the foundation for the methylation analysis of FPS, SS, and SE and the further studying of the mechanism expression regulation in E. Senticosus.

Lack of Aberrant Methylation in an Adjacent Area of Left-Sided Colorectal Cancer

PURPOSE: The molecular nature and the rate-limiting step of epigenetic field defects in the evolution of left-sided colorectal cancer (LCA) remain uncertain. MATERIALS AND METHODS: The methylation status of 27 candidate field defect markers, six classic CpG island methylator phenotype (CIMP) markers, and LINE-1 were determined in LCA and adjacent normal mucosas (ADJs) from 33 LCA patients and in left normal colorectal mucosa (LNM) from 33 age- and sex-matched controls. Hotspot mutation analyses in KRAS codons 12 and 13 and BRAF V600E were performed by genomic PCR and pyrosequencing using DNA extracted from endoscopically biopsied tissues. RESULTS: Among the 27 candidate genes tested, we confirmed 15 differentially methylated genes in cancer (15 DMGs; ER, SFRP1, MYOD1, MGMT, CD8a, SPOCK2, ABHD9, BNIP3, IGFBP3, WIF1, MAL, GDNF, ALX4, DOK5, and SLC16A12) in comparison to ADJ samples. We further compared the methylation status of 15 DMGs of ADJs to LNM and found only methylation levels of SLC16A12 in ADJs of LCA patients to be significantly higher than that in LNM (17.3% vs. 11.5%, p=0.002). Based on the CIMP, no significant differences in methylation levels of the 15 DMGs were found between ADJs in CIMP positive LCA cases and those without CIMP. In mutation analyses, no mutation was found in ADJs, while significant KRAS mutations (6/33, 18%) were noted in LCA samples. CONCLUSION: Epigenetic field defect marked by aberrant methylation is uncommon in normal-appearing ADJs of LCA, indicating the critical rate-limiting change of methylation is likely to occur with morphological alterations in the evolution of LCA.

HOTAIR Long Non-coding RNA: Characterizing the Locus Features by the In Silico Approaches

HOTAIR is an lncRNA that has been known to have an oncogenic role in different cancers. There is limited knowledge of genetic and epigenetic elements and their interactions for the gene encoding HOTAIR. Therefore, understanding the molecular mechanism and its regulation remains to be challenging. We used different in silico analyses to find genetic and epigenetic elements of HOTAIR gene to gain insight into its regulation. We reported different regulatory elements including canonical promoters, transcription start sites, CpGIs as well as epigenetic marks that are potentially involved in the regulation of HOTAIR gene expression. We identified repeat sequences and single nucleotide polymorphisms that are located within or next to the CpGIs of HOTAIR. Our analyses may help to find potential interactions between genetic and epigenetic elements of HOTAIR gene in the human tissues and show opportunities and limitations for researches on HOTAIR gene in future studies.

Classification of Colon Cancer Patients Based on the Methylation Patterns of Promoters

Diverse somatic mutations have been reported to serve as cancer drivers. Recently, it has also been reported that epigenetic regulation is closely related to cancer development. However, the effect of epigenetic changes on cancer is still elusive. In this study, we analyzed DNA methylation data on colon cancer taken from The Caner Genome Atlas. We found that several promoters were significantly hypermethylated in colon cancer patients. Through clustering analysis of differentially methylated DNA regions, we were able to define subgroups of patients and observed clinical features associated with each subgroup. In addition, we analyzed the functional ontology of aberrantly methylated genes and identified the G-protein-coupled receptor signaling pathway as one of the major pathways affected epigenetically. In conclusion, our analysis shows the possibility of characterizing the clinical features of colon cancer subgroups based on DNA methylation patterns and provides lists of important genes and pathways possibly involved in colon cancer development.

Biomarkers and clinicopathologic features of CpG island methylator phenotype lung cancer / 第二军医大学学报

Objective To compare the sensitivity and specificity of the new CpG island methylation phenotype (CIMP) selection marker with the classical CIMP selection marker, and to analyze the clinical pathological features of CIMP lung cancer. Methods Genomic DNA was extracted from 50 cases of lung cancer tissues and the corresponding adjacent normal lung tissues in Changhai Hospital. Methylation statues of new CIMP selction marker (SHISA3, CTSL1, C1ORF103 and TMEM200B) and the classical CIMP selection markers (CACNA1G, IGF2, NEUROG1 and RUNX3) were determined by methylation specific PCR, and the results were analyzed by SPSS software. Results Notable methylation was found in the lung caner tissues, with the methylation levels of the eight studied genes in the lung cancer tissues being significantly higher than those in the adjacent tissues (P=0.014). RUNX3 gene methylation was significantly related to lymph node metastasis and PS score (P=0.017, P=0.018). The methylation rate in lung cancer patients aged over 60 was significantly higher than those aged younger than 60 years old (P=0.031). Smoking showed significant influence on CTSL1 gene methylation (P=0.018). Conclusion CpG island methylation phenotypic lung cancer has unique clinical pathological features. Both the new and classic CIMP markers are of high sensitivity and specificity for the diagnosis of lung cancer.

Effect of folate deficiency on experimental colitis in mice induced by dextran sodium sulfate / 中华消化杂志

Objective To investigate whether folate deficiency cause high expression level of interferon gamma (IFN-γ) resulted from IFN-γ gene ( IFNG) hypomethylation and then promote the pathogenesis and development of ulcerative colitis (UC ) in a dextran sulfate sodium (DSS )-induced experimental colitis model in mice .Methods A total of 24 female BALB/c mice were divided into four groups ,six mice in each group , including folate deficient/DSS+ group , standard diet/DSS+ group , standard diet/DSS - group and folate deficient/DSS- group .At the beginning of the sixth week since fed , the mice of model groups were treated with 5% DSS to establish experimental colitis .By the end of the sixth week ,disease activity index (DAI) of colitis and histological changes were evaluated .The folate level of peripheral blood serum of mice were detected by enzyme-linked immunosorbent assay (ELISA ) . The expression of IFN-γ in colonic mucosa of mice was examined by immunohistochemistry . The methylation level of CpG island in the promoter region of IFNG was determined by methylation specific polymerase chain reaction (MSP) .The t test was used for measurement data .Chi square test was performed for comparison between groups of count data . Spearman correlation analysis was used for correlation analysis .Results The folate levels of peripheral blood serum of folate deficiency/DSS+ group and folate deficiency/DSS- group ((2 .70 ± 0 .19) and (2 .80 ± 0 .25)μg/L) were significantly lower than those of standard diet/DSS+ group and standard diet/DSS- group ((13 .62 ± 0 .38 ) and (13 .52 ± 0 .77)μg/L ,t= -63 .33、32 .27 ,both P< 0 .05) ,resepectively .The expression of IFN-γ in colonic mucosa of folate deficiency/DSS+ group and standard diet/DSS+ group were significantly higher than those of folate deficiency/DSS- group and standard diet/DSS- group (χ2 = 22 .18 ,P< 0 .05 ) . And the expression of IFN-γ in colonic mucosa of folate deficiency/DSS+ group was also higher than that of standard diet/DSS+ group (χ2 = 12 .00 ,P< 0 .05) .The expression level of IFN-γ of folate deficiency/DSS+ group and standard diet/DSS+ group was positively correlated with (r=0 .998、0 .953 ,both P<0 .01) .The folate levels of peripheral blood serum of folate deficiency/DSS+ group was negatively correlated with IFN-γexpression level and DAI (r= -0 .880 and -0 .926 ,both P<0 .05) .No abnormal methylation was detected in IFNG promoter CpG island in colonic mucosa tissues of mice of each group . Conclusion In the mice model of DSS induced acute experimental colitis ,folate deficiency may increace the expression of inflammatory factor IFN-γand enhance the inflammation activity of colonic mucosa .

DNA Methylation as Surrogate Marker For Gastric Cancer

Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern.

Expression and promoter methylation of Kras gene in thymic lymphomas induced by ionizing radiation / 吉林大学学报(医学版)

Objective To study the changes of mRNA and protein expressions of Kras gene in thymic lymphomas induced by ionizing radiation,and to detect the methylation of CpG islands in promoter region of Kras gene,then to investigate the mechanisms for the occurrence of radiation carcinogenesis.Methods The thymic lymphoma models of BALB/c mice were made by X-ray irradiation,then the total RNA was extracted,cDNA was synthesized and the total protein was extracted from both thymic lymphoma tissue and normal thymus tissue;the mRNA and protein expressions of Kras gene in thymic lymphoma tissue and normal thymus tissue were detected by RT-PCR and Western blotting method, and the methylation of CpG islands in promoter region of Kras gene was detected by bisulfite sequencing PCR. Results The mRNA expression level of Kras gene in thymic lymphoma tissue was significantly higher than that in normal thymus tissue(P<0.01).The protein expression level in thymic lymphoma tissue was about 1.41 times higher than that in normal thymus tissue;4 CpG sites were methylated detected by bisulfite sequencing PCR in normal thymus tissue, however, 1 CpG site was methylated in thymic lymphoma tissue,the CpG islands in promoter region of Kras gene were demethylation state in thymic lymphoma. Conclusion Ionizing radiation can cause the changes of mRNA and protein expression levels of Kras gene in thymic lymphoma tissue by demethylation state of Kras gene,eventually lead to the occurrence of tumor;it might be one of the mechanisms for the occurrence of radiation carcinogenesis.

Restrição no consumo de sódio durante a gestação é responsável pelo baixo peso ao nascimento e pela resistência à insulina da prole na idade adulta: estudo do mecanismo epigenético por metilação do DNA / Sodium intake restriction during pregnancy is responsible for low birth weight and the insulin resistance of offspring in adulthood: a study of epigenetic mechanism by DNA methylation

Sabe-se que algumas alterações nutricionais maternas durante o período perinatal estão associadas com doenças metabólicas na vida adulta das proles, tais como diabetes melito tipo 2, resistência à insulina, obesidade e hipertensão arterial. O período da gestação em que estas alterações nutricionais influenciam a prole na idade adulta ainda não está elucidado. Modificações epigenéticas têm sido propostas como mecanismos responsáveis por estas desordens metabólicas. Ratas Wistar de doze semanas de idade foram alimentadas com dieta com conteúdo baixo (HO - 0,15% NaCl) ou normal (NR - 1,3% NaCl) de sódio desde o primeiro dia de gestação até o nascimento da prole ou HO durante a primeira (HO10) ou segunda (HO20) metade da gestação. O peso corpóreo e a ingestão de água e ração foram avaliados semanalmente durante a gestação. Teste de tolerância à insulina (ITT) e à glicose (GTT) e HOMA-IR foram realizados nas proles adultas. Expressão gênica por qRT-PCR e metilação do DNA na região promotora dos genes foram mapeadas utilizando tratamento com bissulfito de sódio e avaliadas por pirosequenciamento. O ganho de peso materno foi menor no HO e HO20 na terceira semana de gestação em comparação com NR e HO10. O peso ao nascimento da prole foi menor em machos e fêmeas dos grupos HO e HO20 em relação ao NR e HO10. O HOMA-IR foi maior nos machos com 12 semanas de idade do grupo HO em comparação com NR e com 20 semanas de idade do grupo HO10 em comparação com NR e HO20. Nas fêmeas com 12 semanas de idade o HOMA-IR foi maior no HO10 comparado com HO. Os níveis de insulina no soro foram maiores tanto nos machos com 20 semanas de idade do grupo HO10 comparado com NR quanto nas fêmeas com 12 semanas de idade do grupo HO10 comparado com HO. A área sob a curva do GTT indicou intolerância à glicose nos machos do grupo HO. A porcentagem de metilação das ilhas CpG no promotor dos genes de Igf1, Igf1r, Ins1, Ins2 e Insr no fígado de machos e fêmeas neonatais e no...

It is known that some maternal nutritional alterations during pregnancy are associated with metabolic disorders in adult offspring, such as insulin resistance, type 2 diabetes mellitus, obesity and arterial hypertension. The period of pregnancy in which these nutritional alterations influence adult offspring remains uncertain. Epigenetic changes are proposed to underlie these metabolic disorders. Twelve-week-old female Wistar rats were fed a low-salt (LS - 0.15% NaCl) or normal-salt (NS - 1.3% NaCl) diet since the first day of gestation until delivery or LS during the first (LS10) or second (LS20) half of gestation. Body weight, food and water intake were weekly evaluated during gestation. Blood glucose, insulin (ITT) and glucose (GTT) tolerance tests, HOMA-IR were performed in adult offspring. Gene expression and DNA methylation were mapped using bisulfite treatment evaluated by pyrosequencing in the male and female neonates and adult offspring. Weight gain was lower in LS and LS20 dams than in NS and LS10 dams in the third week of pregnancy. Birth weights were lower in male and female LS20 and LS rats compared with NS and LS10 neonates. HOMA-IR was higher in 12-week-old LS males compared with NS and in 20-week-old male LS10 rats compared with NS and LS20 rats. In 12-week-old LS10 females, HOMA-IR was higher than in LS. Serum insulin levels were higher in 20 week-old LS10 male compared with NS rats and in 12-week-old LS10 female compared to LS rats. The area under the curve of GTT indicated glucose intolerance in 12- and 20-week-old LS male. Methylation of CpG islands of the Insr, Igf1, Igf1r, Ins1 and Ins2 genes in liver in neonates male and female offspring and liver, white adipose tissue and muscle in 20-week-old male offspring were influenced by low-salt intake during pregnancy. None of these alterations was identified in 20-week-old females. In conclusion, low-salt diet consumption in the second half of pregnancy can result in low birth weights in the...

Pharmacological Unmasking Microarray Approach-Based Discovery of Novel DNA Methylation Markers for Hepatocellular Carcinoma

DNA methylation is one of the main epigenetic mechanisms and hypermethylation of CpG islands at tumor suppressor genes switches off these genes. To find novel DNA methylation markers in hepatocellular carcinoma (HCC), we performed pharmacological unmasking (treatment with 5-aza-2'-deoxycytidine or trichostatin A) followed by microarray analysis in HCC cell lines. Of the 239 promoter CpG island loci hypermethylated in HCC cell lines (as revealed by methylation-specific PCR), 221 loci were found to be hypermethylated in HCC or nonneoplastic liver tissues. Thirty-three loci showed a 20% higher methylation frequency in tumors than in adjacent nonneoplastic tissues. Correlation of individual cancer-related methylation markers with clinicopathological features of HCC patients (n = 95) revealed that the number of hypermethylated genes in HCC tumors was higher in older than in younger patients. Univariate and multivariate survival analysis revealed that the HIST1H2AE methylation status is closely correlated with the patient's overall survival (P = 0.022 and P = 0.010, respectively). In conclusion, we identified 221 novel DNA methylation markers for HCC. One promising prognostic marker, HIST1H2AE, should be further validated in the prognostication of HCC patients.

CpG Island Methylation According to the Histologic Patterns of Early Gastric Adenocarcinoma

BACKGROUND: Although the importance of aberrant DNA methylation in the development of gastric adenocarcinoma has been described, the mechanism of pathogenesis has not been revealed yet. We quantitatively analyzed methylation of four CpG islands and one repetitive DNA element, according to the histologic features of adenocarcinoma with precursor lesions. METHODS: We divided the cases as adenocarcinoma with intestinal type precursors (type A, n=19 cases) and adenocarcinoma with diffuse type precursors (type B, n=19 cases). We micro-dissected tumor cells and matched non-neoplastic gastric mucosa from the hematoxylin and eosin-stained slides. RESULTS: A total of 20 CpG sites of long interspersed nucleotide element-1 (LINE1), RAR-related orphan receptor alpha (RORA), Kruppel-like factor 7 (KLF7), mutL homolog 1 (MLH1), MINT25, and CD133 were analyzed. Methylation was determined by bisulfate-pyro-sequencing, and hypomethylation of LINE1 and CD133 was noted in the tumors, compared to the levels in the non-neoplastic gastric mucosa (p=0.014 and p=0.015, respectively). A statistically different methylation pattern of CpG sites at CD133 and KLF7 was noted only in type B lesions, compared to that in matched non-neoplastic gastric mucosa (p=0.027 and p=0.043, respectively). CONCLUSIONS: Given that aberrant methylation occurs in a relatively early phase of carcinogenesis, different patterns of methylation may determine the carcinoma phenotype. However, further large-scale study is required to clarify the significance of this difference.

Effects of 5-azacytidine and estradiol on the methylation of CpG motifs and expression of DNA methyltransferase 1 in patients with SLE / 中华皮肤科杂志

Objective To investigate the effects of 5-azacytidine and estradiol on the methylation of CpG motifs and expression of DNA methyltransferasel (DNMT1) mRNA in patients with systemic lupus erythematosus (SLE) and normal controls.Methods Peripheral blood lymphocytes isolated from 12 patients with SLE and 11 normal human controls were stimulated with phytohaemagglutinin for one day followed by additional 3-day culture with or without the presence of 5-azacytidine of 1 μmol/L or estradiol of 30μg/L respectively.Then.the methylation of CpG motifs was detected by flow cytometry using anti-5-methylcytosine antibody,and DNMT1 mRNA expression by real time reverse transcription-PCR Results After treatment with 5-azacytidine,a decrease wag observed in the methylation of CpG motifs, but not in the expression of DNMT1 mRNA in peripheral lymphocytes from patients with SLE (1=18.60,P<0.01;t=1.56.P>0.05) and in those from the normal controls (t=5.63,P<0.01;t=2.17,P>0.05) compared with untreated lymphocytes.Nevertheless,there were no significant changes in the methylation of CpG motifs or expression of DNMT1 mRNA in lymphocytes from patients with SLE (t=1.53,0.93,respectively,both P>0.05) and normal controls (t=1.93,0.11,respectively,both P>0.05) after the treatment with estradiol.Conclusions The methylation of CpG motifs is suppressed efficiently by 5-azacytidine,and the suppression is unlikely to be associated with the decrease of DNMT1 mRNA.Estradiol has no significant impact on the methylation of CpG motifs and expression of DNMT1 mRNA in lymphocytes.

Quantitative analysis of NPTX2 hypermethylation in pancreatic cancer and its application in diagnosis of pancreatic cancer / 第二军医大学学报

Objective: To investigate the methylation level of NPTX2 gene in pancreatic cancer and assess its clinical value in diagnosis of pancreatic cancer. Methods: We detected the mRNA expression and methylation levels of NPTX2 gene by qRT-PCR and quantitative methylation with SYBR Green in 10 primary pancreatic carcinoma tissues and the matched adjacent normal tissues. The methylation levels of NPTX2 gene were then detected and compared using the above-mentioned method in the whole blood of patients with pancreatic ductal adenocarcinoma and healthy volunteers. Results: The relative quantification (RQ) of NPTX2 expression in pancreatic carcinoma tissues was significantly lower than that in the adjacent tissues (0.276±0.263 vs 3.526±3.037, P = 0.001). The result of quantitive methylation indicated that the methylation index (MI) of the carcinoma tissues was significantly higher than that of the adjacent tissues ([9.02±7.52]% vs [1.28±0.98]%, P = 0.003). MI of NPTX2 gene was negatively correlated with RQ value (R= -0.552, P = 0.012). We also found that the MI of the whole blood DNA methylation of patients with pancreatic cancer was significantly higher than that of healthy volunteers ([1.80±1.76]% vs [0.84±0.45]%, P < 0.05). Conclusion: Our findings strongly suggest that NPTX2 gene is aberrantly hypermethyated in pancreatic ductal adenocarcinoma. Detection of MI of NPTX2 gene might be of great value for diagnosis of pancreatic cancer.

Identification of Novel Methylation Markers in Hepatocellular Carcinoma using a Methylation Array

Promoter CpG island hypermethylation has become recognized as an important mechanism for inactivating tumor suppressor genes or tumor-related genes in human cancers of various tissues. Gene inactivation in association with promoter CpG island hypermethylation has been reported to be four times more frequent than genetic changes in human colorectal cancers. Hepatocellular carcinoma is also one of the human cancer types in which aberrant promoter CpG island hypermethylation is frequently found. However, the number of genes identified to date as hypermethylated for hepatocellular carcinoma (HCC) is fewer than that for colorectal cancer or gastric cancer, which can be attributed to fewer attempts to perform genome-wide methylation profiling for HCC. In the present study, we used bead-array technology and coupled methylation-specific PCR to identify new genes showing cancer-specific methylation in HCC. Twenty-four new genes have been identified as hypermethylated at their promoter CpG island loci in a cancer-specific manner. Of these, TNFRSF10C, HOXA9, NPY, and IRF5 were frequently hypermethylated in hepatocellular carcinoma tissue samples and their methylation was found to be closely associated with inactivation of gene expression. Further study will be required to elucidate the clinicopathological implications of these newly found DNA methylation markers in hepatocellular carcinoma.

CpG Islands Detector: a Window-based CpG Island Search Tool

CpG is the pair of nucleotides C and G, appearing successively, in this order, along one DNA strand. It is known that due to biochemical considerations CpG is relatively rare in most DNA sequences. However, in particular subsequences, which are a few hundred to a few thousand nucleotides long, the couple CpG is more frequent. These subsequences, called CpG islands, are known to appear in biologically more significant parts of the genome. The ability to identify CpG islands along a chromosome will therefore help us spot its more significant regions of interest, such as the promoters or 'start' regions of many genes. In this respect, I developed the CpG islands search tool, CpG Islands Detector, which was implemented in JAVA to be run on any platform. The window-based graphical user interface of CpG Islands Detector may facilitate the end user to employ this tool to pinpoint CpG islands in a genomic DNA sequence. In addition, this tool can be used to highlight potential genes in genomic sequences since CpG islands are very often found in the 5' regions of vertebrate genes.

Model of aberrant DNA methylation patterns and its applications in epithelial ovarian cancer / 中华妇产科杂志

Objective To profile methylation alterations of cytosine-phosphate-guanosine islands (CGI)in epithelial ovarian cancer and investigate its applications for finding new candidate tumor markers.Methods Cancer cells were obtained by lager microdissection from 20 tissues of frozen-preserved epithelial ovarian tumors.Primary cultured epithelial cells were isolated from 5 tissues of normal ovaries.Differential methylation hybridization(DMH)based on microarray assay Was conducted using DNA to construct the aberrant DNA methylation pattern of epithelial ovarian cancer.MethyLight was conducted to verify the methylation status of 7 hypomethylated promoter CGI detected by DMH in tumor tissues of 87 patients with epithelial ovarian cancer and 42 patients with benigh ovarian diseases.Results The aberrant DNA methylation pattem of epithelial ovarian cancer were included 182 hypermethylated loci and 64 hypomethylated loci,of which the positive loci located more than 25%arrays were 18 and 31,respectively.The methylation ratio of gene LSM2,EGFLAM and CDKN2A in tissue DNA of patients with epithelial ovarian cancer and benign ovarian diseases Was 11%(10/87)versus 33%(14/42),8%(7/87)versus 21%(9/42),9%(8/87)versus 31%(13/42),respectively,which Was significantly decreased in tissues DNA of ovarian cancer than that from benigh ovarian diseases(P＜0.05).Conclusions The aberrant DNA methylation pattern of epithelial ovarian cancer is important for finding new cancer related genes.The promoter CGI of gene ISM2,EGFIAM and CDKN2A may be Hovel candidate for ovarian cancerspecific hypomethylated tumor markers.

The Relationship between the Methylenetetrahydrofolate Reductase Genotypes and the Methylation Status of the CpG Island Loci, LINE-1 and Alu in Prostate Adenocarcinoma

BACKGROUND: Genetic polymorphism of methylenetetrahydrofolate reductase (MTHFR), in association with the influence of MTHFR upon DNA methylation, may cause differences of the methylation profile of cancer. Thus, we investigated the relationship between the methylation status of prostate adenocarcinoma and the genetic polymorphism of MTHFR. METHODS: We examined 179 cases of prostate adenocarcinoma for determining the genotypes of MTHFR 677 and 1298, the methylation status of 16 CpG island loci and the methylation levels of the LINE-1 and Alu repeats with using polymerase chain reaction/restriction fragment length polymorphism, methylation-specific polymerase chain reaction and combined bisulphite restriction analysis, respectively. RESULTS: There was a higher proportion of the CT genotype of MTHFR 677 in the prostate adenocarcinoma than that in the normal control. The TT genotype of MTHFR 677 showed the highest frequency of methylation in six out of nine major CpG island loci, and these were which were frequently hypermethylated in prostate adenocarcinoma. The CT type showed the lowest methylation levels of LINE-1 and Alu among the MTHFR 677 genotypes. Interestingly, the CC type of MTHFR 1298 demonstrated favorable prognostic factors. CONCLUSIONS: Our study is the first to examine the methylation profile of prostate adenocarcinoma according to the MTHFR genotypes. The differences of the cancer risk, the genomic hypomethylation and the prognosis between the MTHFR genotypes in prostate adenocarcinoma should be further explored.