CDH1 Methylation Research Articles

Droplet digital PCR analysis of CDH13 methylation status in Slovak women with invasive ductal breast cancer

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR− versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

CDH1 methylation analysis in invasive lobular breast carcinomas with and without gene mutation

The proposed role of CDH1 (E-cadherin gene) methylation as a mechanism of gene inactivation in invasive lobular carcinoma (ILC) remains inconclusive. For many years, CDH1 promoter hypermethylation has been regarded as a mechanism for gene inactivation in ILC. However, this assumption has primarily relied on non-quantitative assays, which have reported CDH1 methylation frequencies ranging from 26 to 93% at CpG sites within the island region. Few studies employing quantitative methods and covering CpG island shores, regions of relatively low CpG density situated proximal to conventional promoter CpGs, have been conducted, revealing lower percentages of methylation ranging from 0 to 51%. Therefore, using the quantitative pyrosequencing method, we examined CDH1 methylation in the island region and shores in E-cadherin deficient ILC cases (15 with CDH1 mutation and 22 non-mutated), 19 cases of invasive breast carcinomas non-special type (IBC-NSTs), and five cases of usual ductal hyperplasia (UDH). Our analysis revealed CDH1 methylation frequencies ranging from 3 to 64%, with no significant increase in methylation levels in any group of ILCs (median = 12%) compared to IBC-NST (median = 15%). In addition, considering the poorly studied association between the number of tumor-infiltrating lymphocytes (TILs) and CDH1 methylation in breast cancer, we undertook a thorough analysis within our dataset. Our findings revealed a positive correlation between CDH1 methylation and the presence of TILs (r = 0.5; p-value < 0.05), shedding light on an aspect of breast cancer biology warranting further investigation. These findings challenge CDH1 methylation as a CDH1 inactivation mechanism in ILC and highlight TILs as a potential confounding factor in gene methylation.

Increased CpG methylation at the CDH1 locus in inflamed ileal mucosa of patients with Crohn disease

BackgroundE-cadherin, a major actor of cell adhesion in the intestinal barrier, is encoded by the CDH1 gene associated with susceptibility to Crohn Disease (CD) and colorectal cancer. Since epigenetic mechanisms are suspected to contribute to the multifactorial pathogenesis of CD, we studied CpG methylation at the CDH1 locus. The methylation of the CpG island (CGI) and of the 1st enhancer, two critical regulatory positions, was quantified in surgical specimens of inflamed ileal mucosa and in peripheral blood mononuclear cells (PBMC) of 21 CD patients. Sixteen patients operated on for a non-inflammatory bowel disease, although not normal controls, provided a macroscopically normal ileal mucosa and PBMC for comparison.ResultsIn ileal mucosa, 19/21 (90%) CD patients vs 8/16 control patients (50%) (p < 0.01) had a methylated CDH1 promoter CGI. In PBMC, CD patients with methylated CGI were 11/21 (52%) vs 7/16 controls (44%), respectively. Methylation in the 1st enhancer of CDH1 was also higher in the CD group for each of the studied CpGs and for their average value (45 ± 17% in CD patients vs 36 ± 17% in controls; p < 0.001). Again, methylation was comparable in PBMC. Methylation of CGI and 1st enhancer were not correlated in mucosa or PBMC.ConclusionsMethylation of several CpGs at the CDH1 locus was increased in the inflamed ileal mucosa, not in the PBMC, of CD patients, suggesting the association of CDH1 methylation with ileal inflammation. Longitudinal studies will explore if this increased methylation is a risk marker for colorectal cancer.

The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

Methylation Status of CDH1 Gene in Gastric Biopsy Specimens and Its Association with H. Pylori Infection.

Helicobacter pylori (H. pylori) have been accepted as having an etiologic role in gastro-duodenal diseases as chronic gastritis, peptic ulcer, and gastric carcinoma. Methylation of CGI has been correlated with the tumorigenic process since it can inactivate tumor suppressor genes. CDH1 is a tumor suppressor gene that encodes the E-cadherin protein, which is preserving cell-cell connections. Early stages of gastric carcinogenesis may be affected by the promoter methylation-mediated inactivation of this gene. This study aimed to investigate the methylation status of CDH1 using Methylation-Specific PCR (MSP) technique in clinical suspected patients with H. pylori infection who undergoing upper gastrointestinal endoscopy and correlated it with H. pylori detection by glmM PCR test. Fifty gastric mucosal biopsies were selected from one hundred and five samples included in this study. The detection of H. pylori was performed with the PCR primers specific to glmM gene. Bisulfite modification was done and the methylation status of the CDH1 gene was detected using MSP reaction. H. pylori was detected in 36% (18/50) of study population using glmM gene PCR test, 89% (16/18) of H. pylori positive cases were CDH1 methylated positive (chi-square, p-value=0.002). CDH1 methylation can be present in cancerous and noncancerous gastric mucosa, where 60% (18/30) of CDH1 methylation positive gastric mucosa showed gastritis as an endoscopy finding and gastric cancer in 6% (2/30). There was a significant correlation between and CDH1 methylation positive results and age group (P-value = 0.02). There was no significant correlation between CDH1 methylation positive results and participants gender (p-value=0.431) and clinical symptoms (all P-value > 0.05). This work suggested strong significance association between H. pylori infection and CDH1 methylation.

Dual epigenetic changes in diabetes mellitus-associated pancreatic ductal adenocarcinoma correlate with downregulation of E-cadherin and worsened prognosis.

Diabetes mellitus (DM) is a risk factor for pancreatic ductal adenocarcinoma (PDAC) that promotes the promoter methylation of CDH1. It is still unclear whether DM can exert other epigenetic effects, such as altering microRNA (miR) expression, in PDAC. The expression of miR-100-5p is known to be changed in DM patients and can suppress the expression of E-cadherin. In this study, the correlation between DM status and dual epigenetic changes was evaluated in PDAC specimens from patients who underwent radical surgical resection. A total of 132 consecutive patients with PDAC were clinicopathologically evaluated. E-cadherin and nuclear β-catenin expression was measured using immunohistochemistry. DNA and miRs were extracted from the main tumor site on formalin-fixed paraffin-embedded tissue sections. TaqMan miR assays were applied to assess miR-100-5p expression. Bisulfite modification was conducted on the extracted DNA, which was then subjected to methylation-specific polymerase chain reaction. Immunohistochemistry revealed that decreased E-cadherin expression and increased nuclear β-catenin expression were significantly associated with DM and poor tumor cell differentiation. The presence of long-duration DM (≥3 years) was a significant factor contributing to CDH1 promoter methylation (p < 0.01), while miR-100-5p expression was proportionally correlated with the preoperative HbA1c level (R = 0.34, p < 0.01), but not the duration of DM. The subjects with high miR-100-5p expression and CDH1 promoter methylation showed the highest level of vessel invasion and prevalence of tumor size ≥30 mm. PDAC subjects with dual epigenetic changes showed poorer overall survival (OS) than those with a single epigenetic change. miR-100-5p expression ≥4.13 and CDH1 promoter methylation independently predicted poor OS and disease-free survival (DFS) in the multivariate analysis. OS and DFS worsened in DM subjects with both HbA1c ≥ 6.5% and DM duration ≥3 years. Thus, DM is associated with two modes of epigenetic change by independent mechanisms and worsens prognosis.

Novel urine-based DNA methylation biomarkers for urothelial bladder carcinoma detection in patients with hematuria

ABSTRACT Background Urothelial bladder carcinoma (UBC) is usually detected during work-up for hematuria. Cystoscopy and/or contrast-enhanced imaging are the gold standard tools for UBC diagnosis, despite limited by being invasive, expensive and low yield in small flat tumors. Objectives To assess the diagnostic performance of urine-based DNA methylation of six genes (GATA4, P16, P14, APC, CDH1 and CD99) for UBC detection in patients with hematuria. Patients and methods Voided urine was collected from consecutive patients presented with hematuria for urine cytology and DNA methylation assay of the assigned genes using methylation-specific Polymerase Chain Reaction (PCR). Further assessment by office cystoscopy and imaging with subsequent inpatient cystoscopic biopsy for positive findings was done. The diagnostic characteristics of DNA methylation and urine cytology were assessed based on its capability to predict UBC. Results We included 246 patients in the study with identified macroscopic hematuria in 204 (82.9%) patients. Positive cytology was found in 78 (31.7%) patients. DNA methylation of GATA4, P16, P14, APC, CDH1 and CD99 genes was identified in 127 (51.6%), 52 (21.1%), 117 (47.6%), 106 (43.1%), 90 (36.6%) and 71 (28.9%) patients, respectively. The sensitivity of the assigned genes for UBC detection ranges from 35% (95%CI: 31–39) to 83% (95%CI: 79–87). Optimal specificity (SP) (100%) was noted for P16, APC and CDH1 genes. While for the other genes (GATA4, P14 and CD99), the SP was 95% (95%CI: 92–98), 96% (95%CI: 92–99) and 97% (95%CI: 93–99), respectively. On multivariate logistic regression analysis, all genes exclusively demonstrated independent prediction of UBC. On receiver operator characteristic (ROC) analysis, all tested genes methylation showed superior area under the curve (AUC) when compared to urine cytology. Conclusions We have developed a novel urine-based DNA methylation assay for detection of UBC in patients with hematuria with superior diagnostic performance and independent predictive capacity over urine cytology.

Identification of novel immune-related molecular subtypes and a prognosis model to predict thyroid cancer prognosis and drug resistance.

Background: Thyroid cancer is a common malignant tumor of the endocrine system that has shown increased incidence in recent decades. We explored the relationship between tumor-infiltrating immune cell classification and the prognosis of thyroid carcinoma. Methods: RNA-seq, SNV, copy number variance (CNV), and methylation data for thyroid cancer were downloaded from the TCGA dataset. ssGSEA was used to calculate pathway scores. Clustering was conducted using ConsensusClusterPlus. Immune infiltration was assessed using ESTIMATE and CIBERSORT. CNV and methylation were determined using GISTIC2 and the KNN algorithm. Immunotherapy was predicted based on TIDE analysis. Results: Three molecular subtypes (Immune-enrich(E), Stromal-enrich(E), and Immune-deprived(D)) were identified based on 15 pathways and the corresponding genes. Samples in Immune-E showed higher immune infiltration, while those in Immune-D showed increased tumor mutation burden (TMB) and mutations in tumor driver genes. Finally, Immune-E showed higher CDH1 methylation, higher progression-free survival (PFS), higher suitability for immunotherapy, and higher sensitivity to small-molecule chemotherapeutic drugs. Additionally, an immune score (IMScore) based on four genes was constructed, in which the low group showed better survival outcome, which was validated in 30 cancers. Compared to the TIDE score, the IMScore showed better predictive ability. Conclusion: This study constructed a prognostic evaluation model and molecular subtype system of immune-related genes to predict the thyroid cancer prognosis of patients. Moreover, the interaction network between immune genes may play a role by affecting the biological function of immune cells in the tumor microenvironment.

Association between CDH1 methylation and esophageal cancer risk: a meta-analysis and bioinformatics study

ABSTRACT Objective The aim is to evaluate the association of CDH1 methylation with esophageal cancer (EC) risk. Methods The PubMed, Embase, Web of Science, and Cochrane Library databases were systematically searched to identify relevant articles. Pooled odds ratios (ORs) with 95% confidence interval (CI) were estimated using the fixed- or random-effects models. The pooled sensitivity and specificity were calculated to assess the diagnostic value of CDH1 methylation for EC. The results of the meta-analysis were validated using The Cancer Genome Atlas and Gene Expression Omnibus databases. Results Thirteen studies consisting of 1,633 samples were included. A high CDH1 methylation was significantly associated with an increased risk of EC (OR = 10.40, 95% CI = 6.29–17.18). Furthermore, CDH1 methylation status was related to tumor status, lymph node status, and metastasis. For the diagnosis of EC, the pooled sensitivity and specificity of CDH1 methylation were 0.57 (95% CI = 0.39–0.74) and 0.89 (95% CI = 0.81–0.94), respectively. Bioinformatics analysis showed that CDH1 methylation occurred more frequently in EC tissues than in normal controls, in good agreement with the results of the meta-analysis. Conclusion A significant association was found between CDH1 methylation and EC risk. We therefore suggest that CDH1 methylation can serve as a promising diagnostic marker for EC.

The miR-23a/27a/24-2 cluster promotes postoperative progression of early-stage non-small cell lung cancer

Even with optimal surgery, many early-stage non-small cell lung cancer (NSCLC) patients die of recurrence. Unfortunately, there are no precise predictors for postoperative recurrence in early-stage NSCLC, and the recurrence mechanism is still unclear. In this study, we found that simultaneous overexpression of all miRNAs in the miR-23a/27a/24-2 cluster was closely associated with postoperative recurrence, β-catenin upregulation and promoter methylation of p16 and CDH13 in early-stage NSCLC patients. In addition, in vitro and in vivo experiments show that overexpression or inhibition of all miRNAs in the miR-23a/27a/24-2 cluster significantly stimulated or inhibited NSCLC cell stemness, tumorigenicity and metastasis. Furthermore, we demonstrated that the miR-23a/27a/24-2 cluster miRNAs activated Wnt/β-catenin signaling by targeting their suppressors and stimulated promoter methylation-induced silencing of p16 and CDH13 by affecting DNA methylation-related genes expression. Our findings suggest that simultaneous high expression of all miRNAs in the miR-23a/27a/24-2 cluster represents a new biomarker for predicting postoperative recurrence in early-stage NSCLC. The miR-23a/27a/24-2 cluster miRNAs stimulate early-stage NSCLC progression through simultaneously stimulating Wnt/β-catenin signaling, and promoter methylation-induced tumor suppressor genes silencing. In addition, simultaneous inhibition of all miRNAs in the miR-23a/27a/24-2 cluster may be a useful strategy for treatment of early-stage NSCLC recurrence.

Hypermethylation of the RASSF1A gene promoter as the tumor DNA marker for nasopharyngeal carcinoma.

RASSF1A is a tumor suppressor gene. The methylation of RASSF1A has been reported to be associated with nasopharyngeal tumorigenesis. However, the heterogeneity was high among different studies. A meta-analysis was performed to evaluate the value of RASSF1A methylation for the diagnosis and early screening of nasopharyngeal carcinoma. Relevant articles were identified by searching the MEDLINE database. Frequency and odds ratio (OR) were applied to estimate the effect of CDH-1 methylation based on random-/fixed-effect models. The meta-analysis was performed by using MedCalc® software. Subgroup analyses were performed by test method, ethnicity, and source of nasopharyngeal carcinoma samples to determine likely sources of heterogeneity. A total of 17 studies, including 1688 samples (1165 nasopharyngeal carcinoma samples, and 523 from non-cancerous samples) were used for the meta-analysis. The overall frequencies of RASSF1A methylation were 59.68% and 2.65% in case-group and control-group, respectively. By removing the poor relative studies, the heterogeneity was not observed among the studies included. The association between RASSF1A gene methylation and the risk of nasopharyngeal carcinoma was also confirmed by calculating the OR value of 30.32 (95%CI = 18.22-50.47) in the fixed-effect model (Q = 16.41, p = 0.36,I2 = 8.62, 95% CI = 0.00-45.27). Additionally, the significant association was also found between the methylation of the RASSF1A gene and the subgroups. This is the first meta-analysis that has provided scientific evidence that the methylation of RASSF1A is the potential diagnosis, prognosis, and early screening biomarker for nasopharyngeal carcinoma.

Intratumor Epigenetic Heterogeneity-A Panel Gene Methylation Study in Thyroid Cancer.

BackgroundThyroid cancer (TC) is the most common endocrine malignancy, and the incidence is increasing very fast. Surgical resection and radioactive iodine ablation are major therapeutic methods, however, around 10% of differentiated thyroid cancer and all anaplastic thyroid carcinoma (ATC) are failed. Comprehensive understanding the molecular mechanisms may provide new therapeutic strategies for thyroid cancer. Even though genetic heterogeneity is rigorously studied in various cancers, epigenetic heterogeneity in human cancer remains unclear.MethodsA total of 405 surgical resected thyroid cancer samples were employed (three spatially isolated specimens were obtained from different regions of the same tumor). Twenty-four genes were selected for methylation screening, and frequently methylated genes in thyroid cancer were used for further validation. Methylation specific PCR (MSP) approach was employed to detect the gene promoter region methylation.ResultsFive genes (AP2, CDH1, DACT2, HIN1, and RASSF1A) are found frequently methylated (>30%) in thyroid cancer. The five genes panel is used for further epigenetic heterogeneity analysis. AP2 methylation is associated with gender (P < 0.05), DACT2 methylation is associated with age, gender and tumor size (all P < 0.05), HIN1 methylation is associated to tumor size (P < 0.05) and extra-thyroidal extension (P < 0.01). RASSF1A methylation is associated with lymph node metastasis (P < 0.01). For heterogeneity analysis, AP2 methylation heterogeneity is associated with tumor size (P < 0.01), CDH1 methylation heterogeneity is associated with lymph node metastasis (P < 0.05), DACT2 methylation heterogeneity is associated with tumor size (P < 0.01), HIN1 methylation heterogeneity is associated with tumor size and extra-thyroidal extension (all P < 0.01). The multivariable analysis suggested that the risk of lymph node metastasis is 2.5 times in CDH1 heterogeneous methylation group (OR = 2.512, 95% CI 1.135, 5.557, P = 0.023). The risk of extra-thyroidal extension is almost 3 times in HIN1 heterogeneous methylation group (OR = 2.607, 95% CI 1.138, 5.971, P = 0.023).ConclusionFive of twenty-four genes were found frequently methylated in human thyroid cancer. Based on 5 genes panel analysis, epigenetic heterogeneity is an universal event. Epigenetic heterogeneity is associated with cancer development and progression.

Decreased promoter methylation of E-cadherin gene due to overexpression of TET enzyme by vitamin C in MCF-7 cell line

BackgroundE-cadherin (CDH1) is a well-known tumor suppressor gene. The loss of CDH1 gene function caused by genetic or epigenetic alterations may consequently result in tumorigenesis. Vitamin C is an essential factor for epigenetic regulation and its great furnished potential in epigenetic therapy in cancer patients. ObjectiveThe present study aims to evaluate the effect of vitamin C on promoter methylation of the CDH1 gene and its possible correlation with the expression of CDH1 in the MCF-7 cell line. MethodsCDH1 expression was studied by reverse transcription-polymerase chain reaction (RT–PCR) analysis in MCF-7 cell line that was treated with different concentrations of vitamin C. TET enzyme activity was measured by ELISA. Promoter methylation of CDH1 was studied by Methylation-Sensitive High-Resolution Melting (MS-HRM) analysis of bisulfite-modified DNA. ResultsOur results showed that vitamin C increase not only TET enzyme activity but also TET gene overexpressed. In the following, cause to CDH1 gene promoter demethylation and upregulation of CDH1 gene in MCF-7 cell line. ConclusionCancer cells may respond to vitamin C induces CDH1 expression by promoter demethylation due to TET enzyme overexpression.

Evaluation of molecular changes in the mucous membrane close to the resection margin after surgical treatment for gastric cancer

Objective: Secretion of the changes characterizing genetic instability of the gastric mucosa when having esophagus cancer.Methods: 64 patients having esophagus cancer (main group) and 50 patients having gallstone disease were examined. They were undergoing treatment in the hospital No. 1 of the I.M. Sechenov First Moscow State Medical University. In all cases molecular tumor markers in the gastric mucosa after operation and biopsy were examined.Results: Esophagus cancer turned out to be characterized by high frequency of RASSF1А and MLH1 genes methylation and high hTERT, MMP7 and BIRC5 genes expression, and also by high telomerase activity. There was also high frequency of TUSC3, CDH1 and DAPK genes methylation inside gastric mucosa approached to the edge of resection.Conclusions: The study of the frequency of TUSC3, CDH1, DAPK, RASSF1А and MLH1 genes methylation and telomerase activity can be used as clinical markers defining changes in gastric mucosa after its resection concerning cancer.

Promising aberrant DNA methylation marker to predict gastric cancer development in individuals with family history and long-term effects of H. pylori eradication on DNA methylation.

It remains unknown whether individuals with a family history (FH) of gastric cancer (GC) are associated with aberrant DNA methylation. The aim of this study was to investigate the association between aberrant DNA methylation and FH of GC. Using quantitative MethyLight assay, MOS, miR124a-3, NKX6-1, EMX1, CDH1, and TWIST1 methylation levels in the noncancerous gastric mucosa was compared between subjects with and without FH based on GC and Helicobacter pylori (Hp) infection. Changes in the methylation levels were evaluated over time after Hp eradication. In Hp-positive GC patients, MOS (P < 0.001), CDH1 (P < 0.001), and TWIST1 (P = 0.004) methylation were decreased in subjects with FH (n = 64) than in those without FH (n = 58). In Hp-positive controls, MOS methylation was lower in subjects with FH (n = 73) than in those without FH (n = 50) (P = 0.042), while miR124a-3 (P = 0.006), NKX6-1 (P < 0.001), and CDH1 (P < 0.001) methylation were higher in subjects with FH. CDH1 methylation constantly decreased from 2years in GC patients and 3-4years in controls after Hp eradication (all P < 0.001). A persistent decrease in methylation over time was not observed in other genes after eradication. The methylation of MOS and CDH1 provided an association between aberrant DNA methylation and gastric carcinogenesis in FH of GC, a useful marker for GC risk in individuals with FH. Furthermore, CDH1 methylation decreased after Hp eradication.

Relationship between Methylation of FHIT and CDH13 Gene Promoter Region and Liver Cancer.

In order to demonstrate the relationship between methylation of fragile histidine triad (FHIT) and T-cadherin/H-cadherin (CDH13) genes and liver cancer, the methylation status of FHIT and CDH13 was detected in healthy individuals and in Mongolian and Han patients with liver cancer. The phenol-chloroform method was used to extract genomic DNA. The methylation specific polymerase chain reaction method was applied to detect the methylation status of FHIT and CDH13. The relationship between smoking and alcohol consumption and gene (FHIT and CDH13) methylation was analyzed. There was significant difference in methylation rate of FHIT (72.67%, 34.67%) and CDH13 (72.0%, 28.0%) between liver cancer patients and healthy individuals of Mongolian descent (P<0.05), as well as that of FHIT (68%, 30.67%) and CDH13 (64%, 26%) between liver cancer patients and healthy individuals of Han individuals (P<0.05). There was also a relationship between smoking and drinking and the methylation of FHIT and CDH13 (P<0.05). Thus, the methylation of FHIT and CDH13 had a relationship with liver cancer incidence. Smoking and alcohol ingestion may promote the methylation of FHIT and CDH13.

Effect of folic acid and vitamin E on promoter DNA methylation and expression of TGF-β1, ESR-1 and CDH-1 in the uterus of STZ-induced diabetic rats

The present study is the first attempt made to investigate the effects of diabetes on expression and promoter DNA methylation of TGF-β1, ESR-1, and CDH-1 genes and also the effects of folic acid (FA) and vitamin E (Vit E) supplementations on improving diabetes mellitus. STZ-induced diabetic rats were treated with Vit E (200 mg/kg/day) and FA (25 mg/kg/day) for 8 weeks and expression and DNA methylation of TGF-β1, ESR-1, and CDH-1 genes in uterus were analysed. Data indicated that diabetes increases the expression of TGFβ-1 and ESR-1 and decreases CDH-1 expression and TGFβ-1 promoter methylation in the uterus of rats. Vit E and FA improved the negative effects of diabetes by decreasing the expression of TGFβ-1 and ESR-1 and increasing that of CDH-1 in diabetic rats. In conclusion, these findings emphasise that Vit E and FA supplementations could improve negative effects caused by diabetes on uterus function and fertility in diabetic rats.

E-Cadherin, NFATC3, and PLP2 Are Differentially Methylated in Multiple Cancers.

It is well documented that cancer cells have abnormal methylation patterns often caused by faulty methylating machinery. Specifically, E-cadherin, NFATC3, and PLP2 are 3 genes known to be aberrantly methylated in cancer cells. These genes are well documented for their role in signaling pathways involved with cell proliferation, adhesion, migration, and other signs of tumor progression. Therefore, changes in gene expression of CDH1, NFATC3, and PLP2 due to aberrant methylation can lead to profound changes in cellular function and tumor formation. In order to ensure that previous in vitro and in vivo methylation studies match what is observed in the clinic, we utilized a bioinformatics approach to complete an extensive analysis of methylation patterns of these 3 genes, analyzing over 5000 patient samples, across all cancers for which both normal and tumor tissues were available. Specifically, we analyzed overall and site-specific methylation patterns, at CpG islands and shores, of all 3 genes across 14 cancer types. Furthermore, we compared these methylation levels in normal and tumor samples of both matched and unmatched patient samples in order to determine any differences between groups. Finally, we examined whether an aberrant DNA methyltransferase, DNMT3B7, known to be expressed in cancer cells and to alter methylation patterns in vitro correlated with altered overall and site-specific methylation of CDH1, NFATC3, and PLP2 in these patient samples. Our results indicate that methylation patterns of CDH1 and NFATC3 were unexpectedly varied across tumors, contrary to previous studies performed in vitro, while PLP2 showed the expected hypomethylation pattern in tumor tissues. We also observed some correlation between DNMT3B7 expression and methylation patterns of these genes, but patterns were inconsistent. Taken together, these results emphasize the necessity for in vivo and patient studies rather than a complete reliance on in vitro data and provide multiple areas of future research.

E-Cadherin Downregulation is Mediated by Promoter Methylation in Canine Prostate Cancer.

E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, CDH1 methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of CDH1 promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of CDH1 silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the CDH1 promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified CDH1 promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented CDH1 hypermethylation and after 5-Aza-dC treatment, a decreased CDH1 methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and CDH1 hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.

Effects of high progesterone in in-vitro fertilization cycle on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window.

High progesterone is associated with low implantation rate. Our previous study demonstrated that DNA methylation in endometrium was increased in women with high progesterone in IVF cycles. However, the DNA methylation status is still not yet confirmed, and how it affects endometrial receptivity in high progesterone is still unknown. Current study investigated the effects of high progesterone on DNA methylation and gene expression of adhesion molecules on endometrium during implantation window. A cohort study included 20 women with high progesterone (HP) and 20 with normal progesterone (NP) on the day of human chorionic gonadotropin (hCG) administration after controlled ovarian hyperstimulation in IVF cycle. Endometrial tissues were collected on the 7th day after hCG administration. Immunohistochemical staining of DNA methyltransferases (DNMT1 and DNMT3B) and adhesion molecules (MUC1, CDH1 and CTNNB1) were performed. Methylation of MUC1, CDH1, and CTNNB1 promoter regions was detected by Sequenom MassARRAY or bisulfite sequencing PCR. RT-qPCR was used to quantify mRNA expression levels, and correlation of methylation and gene expression level of the adhesion molecules were determined. DNMT3B, but not DNMT1, in nucleus of luminal and glandular epithelial cells in HP group was significantly higher than that in NP group. Promoter regions of CDH1 and CTNNB1, but not MUC1, in endometrium of HP group were hypermethylated. Protein and mRNA expression of MUC1, CDH1, and CTNNB1 in endometrium of HP group was significantly lower than that in NP group. Level of DNA methylation was negatively correlated with the gene expression of CDH1 and CTNNB1, but not MUC1. DNA hypermethylation and low expression of adhesion molecules on endometrium were associated with high progesterone during implantation window, which may contribute to the underlying epigenetic mechanism in the failure of IVF treatment.