Frequency Of Promoter Methylation Research Articles

Aberrant hypermethylation induced downregulation of antisense lncRNA STXBP5-AS1 and its sense gene STXBP5 correlate with tumorigenesis of glioma

AimsThe expression of antisense lncRNA STXBP5-AS1 and its sense gene STXBP5 were found to be downregulated in glioma by RNA sequencing; however, the function and mechanism of both two genes in the development of glioma have not been studied. Materials and methodsQRT-PCR and western blot were used to determine the transcriptional and translational levels of moleculars. MSP and BSP assays were used to evaluate the methylation status of promoter CpG island. MTT, EdU, flow cytometry, and transwell assays were used to reveal biological effects. The in vivo mice model was used to validate the role of target genes in tumorigenesis. Key findingsThe mRNA and protein expression of STXBP5 was significantly downregulated in glioma tissues and positively correlated with prognosis. STXBP5-AS1 was downregulated in glioma cells and tissues, and associated with tumor size and clinical stages. Both of two genes were significantly restored in cells treatment with 5-Aza. The promoter CpG island of STXBP5/STXBP5-AS1 was hypermethylated in glioma cells, but partially methylated in NHA cells. We found that promoter methylation frequency was significantly higher in glioma tissues. Functionally, overexpression of STXBP5 and STXBP5-AS1 inhibited cell proliferation, migration, and invasion and promoted apoptosis in vitro, whereas depletion of STXBP5 and STXBP5-AS1 showed opposite effects. Both the mRNA and protein expression of STXBP5 were positively regulated by STXBP5-AS1. Ectopic expression of STXBP5 and STXBP5-AS1 suppressed tumor formation in vivo. SignificanceOur findings suggested that epigenetically silenced STXBP5-AS1 and STXBP5 might act as novel tumor suppressors of glioma.

Association between Gene Promoter Methylation and Cervical Cancer Development: Global Distribution and A Meta-analysis.

DNA methylation is the main epigenetic event for gene silencing and is associated with carcinogenesis. In this meta-analysis, we evaluated the association between the methylation of the promoter regions of APC, CADM1, CCNA1, CDH1, DAPK, FHIT, HIC1, MAL, MGMT, hMLH1, P16, PAX1, RAR-β, and RASSF1 genes and the risk of cervical cancer development and progression. Overall, 194 eligible studies were identified assessing the associations of promoter methylation status of aforementioned genes with low- and high-grade squamous intraepithelial lesions (LSIL and HSIL) and cervical cancer development. The majority of studies were conducted on Caucasian and Asian populations, whereas rare studies were available on the African population. Promoter methylation frequencies were shown to be significantly higher in LSIL and HSIL cervical cancer cases as compared to control specimens for CADM1, CCNA1, CDH1, DAPK1, FHIT, MAL, P16, PAX1, RAR-β, and RASSF1 genes. A moderate association was found between HIC promoter methylation, whereas APC, MGMT, and hMLH1 promoter methylation was not correlated with cervical cancer development. Promoter methylation could be considered as a noninvasive biomarker for early cervical lesions, making them highly promising targets for a personalized therapeutic approach.

DNA methylation and SNP in IFITM3 are correlated with hand, foot and mouth disease caused by enterovirus 71

ObjectivesTo explore the mechanisms of interferon-induced transmembrane protein 3 (IFITM3) in response to enterovirus-71-associated hand, foot and mouth disease (EV71-HFMD), in terms of DNA methylation, single-nucleotide polymorphism (SNP) genotype and gene expression. MethodsIn total, 120 patients with EV71-HFMD (60 with mild EV71-HFMD and 60 with severe EV71-HFMD) and 60 healthy controls were enrolled in this study. SNP genotype, IFITM3 promoter methylation and mRNA expression of peripheral blood mononuclear cells were examined using the improved multi-temperature ligase detection reaction, quantitative reverse transcriptase polymerase chain reaction and MiSeq, respectively. ResultsThe distribution of methylation in patients with EV71-HFMD was significantly lower compared with healthy controls, and the severe EV71-HFMD group showed the lowest frequency of IFITM3 promoter methylation. The average level of IFITM3 promoter CpG methylation was negatively correlated with IFITM3 mRNA expression, and hypermethylation of several specific CpG units contributed to IFITM3 downregulation. IFITM3 expression and promoter methylation correlated with EV71 infection progression, especially in the severe EV71-HFMD group. Compared with mild cases, genotype GG and the G allele of rs12252 were over-represented in patients with severe EV71-HFMD. ConclusionsIFITM3 methylation status and SNP genotyping may help clinicians to choose the correct treatment strategy for patients with EV71-HFMD.

Promoter hypermethylation regulates vitamin D receptor (VDR) expression in colorectal cancer-A study from Kashmir valley

BackgroundColorectal carcinogenesis (CRC) is a multistep process, involving both genetic and epigenetic modifications of genes involved in diverse pathways ranging from tumor suppression to DNA mismatch repair. PurposeThis study was undertaken to assess the role of promoter methylation of vitamin D receptor (VDR) gene, a transcription factor with myriad biological functions, in relation to its expression and clinicopathological parameters. MethodsTissue specimens were taken from a total of 75 colorectal cancer cases paired with their normal surrounding epithelium and analyzed by Real-time RT-PCR for assessing the expression profile and MS-PCR for analyzing the promoter methylation status of the VDR gene. Blood sample from the same patients was drawn for vitamin D estimation. ResultsThe frequency of promoter methylation in cancerous tissue was 37.33% against 9.33% in normal tissues (p<0.001). The hypermethylated status of VDR promoter showed significantly inverse association with its expression (p=0.008). Furthermore, when compared with the clinical parameters, methylation status of VDR promoter was significantly associated with tumor staging (p=0.008), grading (p<0.001), depth of invasion (p=0.002) and lymph node metastases (p<0.001). Univariate and multivariate analysis indicated patients with increased VDR expression (p<0.001) and decreased methylation status (p=0.012) exhibited longer overall survival. Additionally, serum 25(OH)D3 levels were not significantly associated with any of the patient characteristics. ConclusionOur study, first of its kind from Kashmir, indicated that VDR shows aberrant methylation pattern in CRC with consequent loss in its expression.

Promoter Methylation Status of P53 and E-Cadherin Genes in Gastric Cancer and its Association with Epstein-Barr Virus and Helicobacter Pylori Infections

Objectives: Gastric cancer is one of the leading causes of morbidity and mortality worldwide. As in other cancers, the development and progression of gastric cancer has been attributed to many factors, including infection with Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV), genetic alterations, and epigenetic silencing due to aberrant methylation of tumor suppressor genes. The purpose of this work is to analyze the promoter methylation status of P53 (TP53) and E-cadherin (CDH1) genes and its association with EBV and H. pylori infections in the gastric cancer patients of Grand Casablanca. Methods: In this study, a total of 50 gastric cancer patients were recruited. Methylation in tumor tissues was detected by methylation-specific PCR (MSP), and the clinical relevance was statistically analyzed. Results: Results revealed a methylation of TP53 promoter observed in 36% of gastric carcinoma cases, which was significantly higher than adjacent normal tissue [p?0.0001]. While no methylation of CDH1 promoter was observed. Furthermore, the frequency of TP53 promoter methylation was significantly different with intestinal and diffuse types of gastric cancer [22.3% vs 77.7%; <0.05]. Moreover cases with EBV infection had higher frequencies of TP53 methylation [p<0.05], while no significant correlation between H. pylori and TP53 methylation was observed. Conclusion: Thus; the present data suggest a vital role of epigenetic alteration of TP53 in the causation and development of gastric cancer, especially Epstein-Barr virus-associated gastric cancer in our population. Keywords: Epstein-Barr virus, CDH1, gastric cancer, methylation, TP53, helicobacter pylori

Infratentorial IDH-mutant astrocytoma is a distinct subtype.

Diffuse IDH-mutant astrocytic tumors are rarely diagnosed in the cerebellum or brainstem. In this multi-institutional study, we characterized a series of primary infratentorial IDH-mutant astrocytic tumors with respect to clinical and molecular parameters. We report that about 80% of IDH mutations in these tumors are of non-IDH1-R132H variants which are rare in supratentorial astrocytomas. Most frequently, IDH1-R132C/G and IDH2-R172S/G mutations were present. Moreover, the frequencies of ATRX-loss and MGMT promoter methylation, which are typically associated with IDH mutations in supratentorial astrocytic tumors, were significantly lower in the infratentorial compartment. Gene panel sequencing revealed two samples with IDH1-R132C/H3F3A-K27M co-mutations. Genome-wide DNA methylation as well as chromosomal copy number profiling provided further evidence for a molecular distinctiveness of infratentorial IDH-mutant astrocytomas. Clinical outcome of patients with infratentorial IDH-mutant astrocytomas is significantly better than that of patients with diffuse midline gliomas, H3K27M-mutant (p < 0.005) and significantly worse than that of patients with supratentorial IDH-mutant astrocytomas (p = 0.028). The presented data highlight the very existence and distinctiveness of infratentorial IDH-mutant astrocytomas that have important implications for diagnostics and prognostication. They imply that molecular testing is critical for detection of these tumors, since many of these tumors cannot be identified by immunohistochemistry applied for the mutated IDH1-R132H protein or loss of ATRX.

DNA methylation patterns of RAR-β2 and RASSF1A gene promoters in FNAB samples from Greek population with benign or malignant breast lesions.

Promoter hypermethylation is common in Breast Cancer (BC) with studies mainly in histological specimens showing frequent methylation of tumor suppressor genes (TSGs) compared with normal tissues. The aim of this study was to estimate the frequency of promoter methylation of RAR-β2 and RASSF1A genes in breast FNAB material aiming to evaluate the methylation status of these two genes as biomarker for detecting BC in Greek population. FNAB material from 104 patients was collected for cytological evaluation and epigenetic analysis. DNA was extracted and subjected to bisulfite conversion. A methylation-specific PCR was carried out and the final products were separated with electrophoresis in 2% agarose gels. From 104 samples, RASSF1A hypermethylation was observed in 78 (75%) and RAR-β2 hypermethylation in 64 (61.6%). 84% and 78% of the cases diagnosed with breast malignancy (n = 50) were methylated for RASSF1A and RAR-β2, respectively. Methylated RASSF1A and RAR-β2 were also detected in 88.3% and 76.5% in samples diagnosed as suspicious for malignancy (n = 17) and in 57.2% of samples diagnosed with atypia (n = 14). The Odds Ratio for breast malignancy was 4.545 in patients with RASSF1A hypermethylation and 9.167 in patients with RAR-β2 hypermethylation underlying their promoter's methylation positive correlation with breast malignancy. To optimize the sensitivity and specificity of this epigenetic setting, more TSGs related to BC should be gradually imported in our evaluated methylation panel and be validated in a larger study sample with the aim that the obtained epigenetic profiles will provide clinicians with valuable tools for management of BC patients in Greece.

Meta-analysis: Assocation between promoter hypermethylation of DAPK (Death-Associated Protein Kinase) and cervical cancer

Purpose: Death-associated protein kinase (DAPK or DAPK1) is an important tumor suppressor protein that involved in the regulation of cell activities. The aberrant methylation of DAPK promoter has been reported in patients with cervical cancer. However, the association between of DAPK1 and cervical cancer was not always unification, in previous studies. Therefore, in current study, a meta-analysis was performed for association of between DAPK gene’s promoter hypermethylated and cervical cancer.&#x0D; Methods: A systematic literature analysis was conducted based on the previous studies published in PubMed, PubMed Central (NCBI), Google by using following keywords: cervical cancer, cervical carcinoma, Methylation, by the end of January, 2018. The association between DAPK promoter methylation and cervical cancer was evaluated by odds ratio (ORs) and 95% confidence intervals (CI). To evaluate the potential sources of heterogeneity, the meta-regression analysis and subgroup analysis were conducted.&#x0D; Results: A total of 21 case-control studies which relevant to the association between DAPK1 gene’s promoter methylation frequency and cervical cancer, that including 1600 cancer cases and 1011 control cases (non-cancerous cases). The analysis results indicated that the characteristic of candidate gene’s promoter methylation increased the cervical cancer risk through the calculation of OR value (OR = 21.25; 95% CI = 8.73 – 52.97; p &lt; 0.001; Random effect model). The association between DAPK1 gene’s promoter hypermethylation was confirmed in all the subgroups analyses, including materials and assays methods, ethnicity. Furthermore, this association is higher in cervical squamous cell carcinoma than cervical adenocarcinoma and is a characteristic of late-stage disease.&#x0D; Conclusion: The hypermethylated DAPK1 gene’s promoter was also one of etiological factor, lead to the cervical tumorigenesis.

The association between the methylation frequency of BRCA1/2 gene promoter and occurrence and prognosis of breast carcinoma: A meta-analysis.

Objectives:Breast cancer susceptibility gene 1/2 (BRCA1/2) is a promising tumor marker in many types of cancer. However, the methylation frequency of BRCA1/2 gene with occurrence risk and survival benefit of patients with breast carcinoma remains controversy. The aim of the present study was to assess the relationship between BRCA1/2 gene promoter methylation and the occurrence and prognosis in breast carcinoma based on a meta-analysis, meanwhile, this article explored the differential expression levels of BRCA1/2 gene promoter methylation in peripheral blood and tumor tissues of breast cancer patients.Methods:Electronic databases (PubMed, Medline, Cochrane Library, and CNKI) were searched up to June 2019. The number of BRCA1/2 promoter methylation-positive and -negative patients in breast carcinoma patients were measured, and hazard ratio (HR) with 95% confidence interval (CI) for the association between BRCA1/2 gene promoter methylation and the prognosis of breast carcinoma patients. Primary end points were presence of breast cancer, overall survival (OS), disease-free survival (DFS). Statistical analysis was performed with STATA 12.0.Results and conclusions:Fifty-eight articles including 19,084 individuals met full eligibility criteria. We observed that the frequency of BRCA1 gene promoter methylation was higher in breast cancer tissues compared with normal tissues, and the prognostic analysis suggested that BRCA1 gene promoter methylation was significantly associated with poor overall survival and poor disease-free survival. This study also verified that there was no statistically significant difference in the methylation frequency of BRCA1 gene promoter between peripheral blood and tumor tissues in breast cancer patients, which suggests that the detection of BRCA1 promoter methylation in peripheral blood may be a non-invasive and rapid way to monitor the occurrence breast cancer.

Association of promoter methylation of RASSF1A and KRAS mutations in non-small cell lung carcinoma in Kashmiri population (India)

BackgroundNon-small cell lung carcinoma (NSCLC) incidence and progression is increasing because of genetic and epigenetic changes. The mutations in the Kirsten rat sarcoma (KRAS) are the most frequently oncogene aberrations in lung carcinoma patients. A candidate tumor suppressor gene (TSG) Ras Association Domain Family 1 Isoform A (RASSF1A), is silenced by promoter hypermethylation in several human malignancies including non-small cell lung carcinoma (NSCLC). We hypothesized that RASSF1A methylation and KRAS mutations may play an important role in NSCLC. MethodsNon-small cell lung carcinoma patients (n = 100) and equal number of healthy controls were assessed for activating KRAS (exon 2) mutations using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and promoter hypermethylation of RASSF1A using methylation specific PCR. ResultsThe frequency of mutations in Kirsten rat sarcoma (KRAS) were found in 31% of NSCLC patients in the Kashmiri population and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. The NSCLC patients in advanced stage reported the higher frequency of mutation in KRAS (exon 2). A significant higher frequency of this mutation was reported in patients with NSCLC (29.16%) who are positive for metastasis (P < 0.03). The frequencies of promoter hypermethylation at Ras Association Domain Family 1 Isoform A (RASSF1A) were 41% in cases and 3% in control samples. The frequency of KRAS mutation and RASSF1A promoter methylation were significantly different between adenocarcinomas (ADC) and squamous cell carcinomas (SCC) patients with NSCLC (P < 0.03). In addition, we reported that NSCLC patients having RASSF1A promoter methylation was significantly associated with smoking (P = 0.01). It was identified that NSCLC patients with RASSF1A promoter region hypermethylation had poorer survival and faster disease progression compared with those without hypermethylation of RASSF1A promoter region (P = 0.0001). The Median survivals among with cases containing promoter region hypermethylation of RASSF1A were 17.20 and 42.13 months for patients without promoter region hypermethylation of RASSF1A and the patients with KRAS mutation with or without hypermethylation of the promoter region of RASSF1A a tumor suppressor gene had poorer survival compared with those patients with wild type KRAS gene, with or without hypermethylation of RASSF1A promoter region. These differences were statistically significant based on Log-rank (Mantel-cox) test (P = 0.0001). The median survivals among patients with mutation in KRAS protooncogene were 16 months and 42 months for NSCLC patients with wild type KRAS gene. ConclusionsThe aberrant RASSF1A gene promoter methylation with the subsequent mutation in KRAS gene (exon 2) plays a significant role in the pathogenesis and disease progression of non-small cell lung carcinoma (NSCLC).

Epigenetic downregulation of desmin in gall bladder cancer reveals its potential role in disease progression

Background & objectives:Gall bladder cancer (GBC) is a fatal neoplasm, with a globally variable incidence rates. To improve the survival rate of patients, a newer set of biomarkers needs to be discovered for its early detection and better prognosis. Our earlier studies on GBC proteomics and whole-genome methylome data revealed expression of desmin to be significantly downregulated with correlated promoter hypermethylation during gall bladder carcinogenesis. Thus, to evaluate desmin as a potential biomarker for GBC, we carried out a detailed follow up study.Methods:Methylation-specific polymerase chain reaction (MS-PCR) (n=17, GBC and n=23, non-tumour control), real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) [n=14, GBC and n=14, adjacent non-tumour (ANT)], immunohistochemistry (n=27, GBC and n=14, non-tumour) and immunoblotting (n=13, GBC and n=13, ANT) were performed in surgically removed gall bladder tissue samples.Results:MS-PCR analysis showed methylation of desmin in 88.23 per cent (15/17) gall bladder tumour samples as compared to non-tumour tissues (39.13%, 9/23). Real-time qRT-PCR analysis revealed a significant downregulation of desmin expression in GBC as compared to ANT tissue. This was further confirmed by western blot, showing reduced expression of desmin protein in GBC, as compared to non-tumour tissue. Immunohistochemical analysis also showed a decreased level of desmin i.e., more than 95 per cent (26/27) in tumour cells compared to non-tumours (35.71%, 5/14).Interpretation & conclusions:The increased frequency of desmin promoter methylation which could be responsible for its significant downregulation, indicates its potential as a candidate biomarker for GBC. This requires further validation in a large group of patients to evaluate its clinical utility.

The Relationship Between Promoter Methylation of RASSF1A Gene and Prognosis of Patients Affected with Breast Cancer in Khuzestan Province

Background DNA methylation is one of the most frequent molecular changes that are associated with human cancers. Tumor suppressor genes are the important targets of hypermethylation in breast cancer and thus may result in cancer by deregulation of cell growth and division.Objectives The purpose of this research was to compare the methylation pattern of the RASSF1A gene in cancerous and normal tissues.Methods Twenty breast cancer patients with known clinicopathologic characteristics and 20 healthy women as control were studied for analysis of the methylation status of the RASSF1A gene promoter by methylation-specific polymerase chain reaction (MS-PCR) technique.Results The findings showed that the frequency of RASSF1A promoter methylation was in 15% of tumor tissues and 5% of normal tissues. These results indicate that there is no statistically significant relationship between the RASSF1A promoter methylation status of tumor tissues and normal tissues (P > 0.05).Conclusions Until now, the evidence for powerful methylation biomarkers is still incomplete and the recognized biomarkers require further validation.

Detection of Gene Promoter Methylation and Protein Expression of p21 in Psoriasis

Abstract Object: The study was aimed to detect the methylation patterns in psoriasis tissues. Methods: We collected 15 psoriasis tissues and 19 uninvolved psoriatic samples from psoriasis patients, and 3 normal skin samples from healthy control, and investigated the methylation levels of p21 gene promoter by pyrosequencing analysis. The expression of p21 protein was detected by immunohischemistry in 38 psoriatic tissues and 16 uninvolved samples from psoriasis patients, and 3 normal samples from healthy control. The differences among the three groups were analyzed by Chi-square test. Results: We found that there was no significant difference in the frequency of p21 gene promoter methylation among the three groups (P=0.204), howerver the p21 protein expression were different among the three groups (P=0.007). Conclusion: This methylation marker maybe not an indicator for the detection and monitoring of psoriasis.

A seven-gene methylation markers panel: An epigenetic predictor of neoadjuvant chemotherapy sensitivity in triple-negative breast cancer

Abstract Background Nowadays anthracycline and taxane-based chemotherapy remains the standard of care for TNBC, with pathological complete response (pCR) rates ranging ∼ 10–30%. pCR in TNBC is associated with better outcomes, while residual disease after neoadjuvant chemotherapy (NACT) have a higher relapse risk and poor prognosis. We have evaluated the utility of DNA methylation markers for TNBC pCR prediction. Methods Epigenetic assessment of DNA extracted from 29 biopsy samples obtained from TNBC patients prior to NACT was performed by genome-wide analysis of DNA methylation by XmaI-RRBS, and CpG dinucleotides differential methylation of which discriminates TNBC samples with different sensitivity to anthracycline and taxane-based NACT were selected. Results Based on XmaI-RRBS results we have developed a seven-gene methylation marker panel (PRKCB, PTGER2, TMEM132D, VGLL4, MYO15B, GMDS, CDO1) predicting TNBC sensitivity to NACT (cisplatin, doxorubicin, paclitaxel) with an area under the ROC curve (AUC) = 0.87 (95% CI = 0.73-1.00). The diagnostic properties of the system were estimated: specificity was 93%, sensitivity - 76% and accuracy - 0.86%. Table: 58P . Gene promoter methylation frequencies in tumors responding and not responding to anthracycline and taxane-based NACT Gene Non-responders, % Responders, % PRKCB 6.3 30.8 PTGER2 56.3 69.2 TMEM132D 87.5 92.3 VGLL4 56.3 38.5 MYO15B 81.3 76.9 GMDS 56.2 53.8 CDO1 43.8 7.7 Conclusions These results show that NACT pCR in TNBC could be accurately predicted with a seven-gene methylation markers panel. Reasonable diagnostic sensitivity and specificity values are achieved only when the markers are evaluated in complex; in separate the differences in gene methylation frequencies between responding and non-responding tumors may be negligible (Table). Further verification of these results on the validating cohort is warranted. Legal entity responsible for the study Research Centre for Medical Genetics. Funding Russian Science Foundation (project No.18-15-00430). Disclosure All authors have declared no conflicts of interest.

The Relationship between the Methylation of Promoter Regions of Tumor Suppressor Genes PTEN and APC with Endometrial Cancer.

Background:Endometrial neoplasms is one of the most typical gynecologic diseases with harmful effects. Promoter hypermethylation is an important mechanism of the inactivation of tumor suppressor genes in endometrial neoplasms. Epigenetic changes of the PTEN and APC genes have shown to be present in various cancers. Therefore, in this study, we have investigated the association between the promoter hypermethylation of PTEN and APC genes with endometrial neoplasms. Methods:For this study, 28 patients with endometrial neoplasms as well as 22 controls were studied. Analysis of the promoter methylation regions of PTEN and APC genes were performed by Methylation-Specific PCR. Results:The frequency of PTEN and APC genes promoter methylation was 28.57% and 17.86% in tumor tissues, and 11.54% and 3.85% in blood samples, respectively. We found a significant relationship between blood and tissue in PTEN methylation (p = 0.0353). Additionally, we determined a closely significant difference between normal tissue and tumor tissue of the PTEN gene (p = 0.0787) and blood and tissue samples of the APC gene in methylated promoter regions (p=0.0623). Furthermore, these results suggest that there is no significant relationship between the promoter methylation of PTEN and APC with clinical characteristics.Conclusion:DNA methylation deficiency is a well known highlighted factor in tumorigenesis, therefore the promoter hypermethylation of PTEN and APC can be indicated as a risk factor in endometrial neoplasms.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.

Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir

Aims:Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic Ser/Thr kinase that participates in cell apoptosis and tumor suppression. DAPK1 is frequently lost in many different tumor types including breast cancer. The aim of this study was to evaluate the promoter methylation status of DAPK1 and a possible correlation with the expression of DAPK1 and standard clinicopathological features in invasive ductal breast carcinoma patients (IDC).Methods:Methylation Specific PCR (MSP) was carried out to investigate the promoter methylation status of DAPK1 from 128 breast cancer patients. The effect of promoter methylation on protein expression was evaluated by immunohistochemistry (n=128) and western blotting (n=56).Results:We found significant difference in DAPK1 promoter methylation frequency among breast tumors when compared with the corresponding normal tissues. Hypermethylation of DAPK1 is significantly correlated with the loss of DAPK1 protein expression (P < .001, rs= -0.361). The loss of DAPK1 protein was significantly associated with estrogen receptor (ER) negativity (p= 0.003), triple negative breast cancer (TNB) (p= 0.024) and advanced tumor stages (P = 0.001). Moreover, age at diagnosis (p= 0.041), tumor stage (p= 0.034), ER negativity (p= 0.004) and TNB cancers (p=0.003) correlated significantly with the hypermethylation of the DAPK1 promoter.Coclusion:This study indicates that DAPK1 is methylated in IDC and promoter hypermethylation could be attributed to silencing of DAPK1 gene expression in breast cancer. Thus, we consider DAPK1 inactivation by promoter hypermethylation likely plays a role in the development and progression of breast cancer.

SFRP2 Promoter Methylation Analysis in Tumor Tissue, Stool, and Plasma DNA of Patients with Colorectal Cancer

Noninvasive method for early detection of human cancers is promising way to provide more quality health service to the population. The serum and stool of colorectal cancer patients often harbor increased free DNA levels, which can potentially be used for cancer detection. Because SFRP2 (secreted frizzled related protein 2) is considered a Wnt inhibitor whose CpGs were frequently hypermethylated in several human cancers, we investigated the frequency of aberrant SFRP2 promoter methylation in primary tumors and serum or stool samples of colorectal cancer patients by MS-PCR. We detected methylation of SFRP2 in 111 of 117 (94.9%) paraffin-embedded colorectal cancer tissues. Fifty-nine of the 68 (86.8%) available serum samples from these cases carried detectable amounts of the methylated SFRP2 promoter. Sixty-one of 68 (89.7%) available stool samples from the same cases carried positive methylation in the promoter region of SFRP2 gene. In contrast, no methylated SFRP2 promoter DNA was detected in serum and stool samples from 60 healthy controls. Only 2 cases of 68 healthy controls showed methylated SFRP2 promoter DNA in their colorectal tissue. But high frequency of methylated SFRP2 gene promoter has no correlation with the clinic-pathologic features. We concluded that SFRP2 methylation analysis appeared to be a noninvasive tumor marker in serum and/or stool DNA

Hypomethylation of mismatch repair genes MLH1 and MSH2 is associated with chemotolerance of breast carcinoma: Clinical significance.

The aim of the study was to understand the importance of mismatch repair genes MLH1 and MSH2 in chemotolerance and prognosis of breast carcinoma (BC). First, the alterations (deletion/methylation/expression) of MLH1 and MSH2 were analyzed in 45 neoadjuvant chemotherapy (NACT)-treated and 133 pretherapeutic BC samples. The chemotolerant BC cells were characterized by treating two BC cell lines MCF-7 and MDA MB 231 with two anthracycline antitumor antibiotics, doxorubicin and nogalamycin. The deletion frequencies were 32% to 38% in MLH1/MSH2 genes and promoter methylation frequencies were 49% to 62% in MLH1 and 41% to 51% in MSH2 in both NACT-treated and pretherapeutic samples. The overall alteration of MLH1 and MSH2 was 58% to 71% in the samples. Reduced messenger RNA (mRNA) and protein expression were found in both the genes and it showed concordance with the molecular alterations. NACT-treated patients showed better prognosis. The chemotherapeutic drug induced increased mRNA/protein expression of the genes in BC cell lines was due to their promoter hypomethylation, as analyzed by quantitative methylation assay. This phenomenon was also evident in NACT-treated BC samples. MLH1/MSH2 genes play a critical role in the development of BC. Hypomethylation of MLH1/MSH2 genes might be important in chemotolerance of the disease.

Decreased Methylation of IFNAR Gene Promoter from Peripheral Blood Mononuclear Cells Is Associated with Oxidative Stress in Chronic Hepatitis B.

Type I interferons (IFNs) play an antiviral effect by binding to type I interferon receptor (IFNAR). Oxidative stress might induce the gene promoter methylation. The purpose of our study was to evaluate the potential relationship between the methylation of IFNAR promoter and the status of oxidative stress in chronic hepatitis B (CHB). The methylation level of the IFNAR promoter in patients with CHB and healthy controls (HCs) was determined by methylation-specific polymerase chain reaction (MS-PCR). The quantitative real-time PCR (RT-qPCR) was used to evaluate the IFNAR mRNA status in peripheral blood mononuclear cells from CHB and HCs. Level of plasma-soluble IFNAR and oxidative stress parameters, including malondialdehyde (MDA) and glutathione (GSH) were determined by enzyme-linked immunosorbent assay (ELISA). The frequency of IFNAR promoter methylation in CHB patients was significantly lower than that of HCs. The IFNAR mRNA level of patients with CHB was higher than HCs. MDA level was higher in CHB patients, whereas GSH level was lower in CHB patients than that of HCs. In CHB patients, plasma MDA level was significantly higher with IFNAR promoter methylation than unmethylation, and soluble IFNAR in the circulation of methylated patients with CHB was decreased than unmethylated patients with CHB. Our results indicated that the IFNAR promoter methylation might have a potential relationship with the status of oxidative stress.