Methylation Status Of Gene Promoter Research Articles

Silencing of MBD2 and EZH2 inhibits the proliferation of colorectal carcinoma cells by rescuing the expression of SFRP.

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal‐to‐amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC‐mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C‐induced amoeboid cells display increased expression of cancer stem‐like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C‐induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C‐driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early‐stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Assessment of DAPK1 and CAVIN3 Gene Promoter Methylation in Breast Invasive Ductal Carcinoma and Metastasis.

Objective Metastasis might be latent or occur several years after primary tumor removal. Currently used methods for detection of distant metastasis have still some limitations. Blood tests may improve sensitivity and specificity of currently used screening procedures. The present study was designed to investigate promoter methylation status of DAPK1 and CAVIN3 genes in plasma circulating free DNA (cfDNA) samples in Iranian invasive ductal carcinoma (IDC) patients. We also investigated association of two gene promoter methylations with breast cancer (BC) and metastatic BC was also assessed. Materials and Methods In this case-control study, MethySYBR assay was performed to determine DAPK1 and CAVIN3 promoter methylation status in breast IDC from 90 patients and 30 controls. Based on clinicopathological information, patient samples subdivided into stage I, II/III and IV groups (each group contained 30 individuals). Results According to the results an increased promoter methylation level of the DAPK1 gene in BC patients was observed. It was found that as disease progressed, the percentage of methylation was changed while it was not significant. Methylation changes in metastatic and non-metastatic BC revealed that methylation levels were significantly increased in metastatic than non-metastatic group. Analysis revealed that promoter methylation of CAVIN3 gene in BC patients was significantly increased. The observed methylation changes from less to more invasive stages were not significant in the CAVIN3 gene. Moreover, promoter methylation was changed in metastatic rather than non-metastatic condition, although it was not significant. ConclusionPromoter hypermethylation of DAPK1 and CAVIN3 genes in plasma are associated with the risk of BC and they can be potential diagnostic biomarkers along with current methods. Additionally, association of aberrant DAPK1 promoter methylation with metastasis suggests its potential usage as a non-invasive strategy for metastatic BC diagnosis.

Methylation degree of metalloproteinase inhibitor RECK gene: Links to RECK protein level and hepatocellular carcinoma in chronic HCV infection patients.

The RECK gene, a tumor suppressor gene, inhibits angiogenesis, invasion, and tumor metastasis. Epigenetic regulation of the RECK gene constitutes a potent approach to the molecular basis of liver malignancy. This study aims to evaluate the promoter methylation status of the RECK gene and its serum level in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) and the potential association of RECK gene methylation with clinical criteria of HCC. One hundred and fifty-five subjects were included (healthy control [55], chronic HCV patients [55], HCV-related HCC patients [45]). The methylation status of the RECK gene promoter and serum RECK level were investigated by methylation-specific PCR and enzyme-linked immunosorbent assay techniques, respectively. RECK gene promoter hypermethylation was recorded in 46.7% of HCC patients, and 10.9% of HCV patients, but not in control subjects (0%). It was related to RECK protein level, varices, edema, ascites, lymph node metastasis, vascular invasion, and the largest diameter of focal lesions. Meanwhile, it was not associated with focal lesionnumber nor distant metastasis of HCC. In conclusion, RECK gene promoter hypermethylation is linked to HCV genotype-4-related HCC. Moreover, different degrees of RECK gene promoter methylation are associated with serum RECK level, lymph node metastasis, and vascular invasion, which could prove its pathogenic role inhepatocarcinogenesis in chronic HCV-infected patients.

Human HAND1 inhibits the conversion of cholesterol to steroids in trophoblasts

Steroidogenesis from cholesterol in placental trophoblasts is fundamentally involved in the establishment and maintenance of pregnancy. The transcription factor gene heart and neural crest derivatives expressed 1 (Hand1) promotes differentiation of mouse trophoblast giant cells. However, the role of HAND1 in human trophoblasts remains unknown. Here, we report that HAND1 inhibits human trophoblastic progesterone (P4) and estradiol (E2) from cholesterol through downregulation of the expression of steroidogenic enzymes, including aromatase, P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1). Mechanically, although HAND1 inhibits transcription of aromatase by directly binding to aromatase gene promoter, it restrains transcription of P450scc by upregulation of the methylation status of P450scc gene promoter through its binding to ALKBH1, a demethylase. Unlike aromatase and P450scc, HAND1 decreases 3β-HSD1 mRNA levels by the reduction of its RNA stability through binding to and subsequent destabilizing protein HuR. Finally, HAND1 suppresses circulating P4 and E2 levels derived from JEG-3 xenograft and attenuates uterine response to P4 and E2. Thus, our results uncover a hitherto uncharacterized role of HAND1 in the regulation of cholesterol metabolism in human trophoblasts, which may help pinpoint the underlying mechanisms involved in supporting the development and physiological function of the human placenta.

Methylation status of genes escaping from X-chromosome inactivation in patients with X-chromosome rearrangements

BackgroundX-chromosome inactivation (XCI) is a mechanism in which one of two X chromosomes in females is randomly inactivated in order to compensate for imbalance of gene dosage between sexes. However, about 15% of genes on the inactivated X chromosome (Xi) escape from XCI. The methylation level of the promoter region of the escape gene is lower than that of the inactivated genes. Dxz4 and/or Firre have critical roles for forming the three-dimensional (3D) structure of Xi. In mice, disrupting the 3D structure of Xi by deleting both Dxz4 and Firre genes led to changing of the escape genes list. To estimate the impact for escape genes by X-chromosome rearrangements, including DXZ4 and FIRRE, we examined the methylation status of escape gene promoters in patients with various X-chromosome rearrangements.ResultsTo detect the breakpoints, we first performed array-based comparative genomic hybridization and whole-genome sequencing in four patients with X-chromosome rearrangements. Subsequently, we conducted array-based methylation analysis and reduced representation bisulfite sequencing in the four patients with X-chromosome rearrangements and controls. Of genes reported as escape genes by gene expression analysis using human hybrid cells in a previous study, 32 genes showed hypomethylation of the promoter region in both male controls and female controls. Three patients with X-chromosome rearrangements had no escape genes with abnormal methylation of the promoter region. One of four patients with the most complicated rearrangements exhibited abnormal methylation in three escape genes. Furthermore, in the patient with the deletion of the FIRRE gene and the duplication of DXZ4, most escape genes remained hypomethylated.ConclusionX-chromosome rearrangements are unlikely to affect the methylation status of the promoter regions of escape genes, except for a specific case with highly complex rearrangements, including the deletion of the FIRRE gene and the duplication of DXZ4.

Prognostic value of methylation status of MGMT gene promoter in patients with glioblastoma after combination treatment.

e14025 Background: The effectiveness of radiation therapy in the classical and stereotactic treatment regimens of the primary glioblastoma multiforme was analyzed and compared. Methods: 87 patients with primary glioblastoma multiforme were hospitalized during the study period. 17 patients of them were in the treatment group (stereotactic radiotherapy, dose per fraction 2 Gy, 60 Gy total dose, CTV 2.0 cm, PTV 0.1 cm - group 1), and 70 patients were in the control group (conventionally fractionated radiotherapy, dose per fraction 2 Gy, 60 Gy total dose, CTV 2.0 cm, PTV 0.5 cm - group 2). Patients of both groups were treated with alkylating agents as the third stage of complex treatment. MGMT methylation was evaluated by pyrosequencing. Results: The use of stereotactic radiation therapy allowed to conduct a full course of treatment without interruption, whereas in the second group, due to the development of adverse reactions, the course was interrupted for 7 patients (10%). The advantage of stereotactic radiation therapy was a less pronounced increase and rapid regression of neurological deficits during treatment, manifested by better local control for 6 months. Hypermethylation of MGMT was detected in 39% of glioblastoma cases and was not detected in non-tumor tissue. The presence of promoter methylation disrupts DNA repair and is associated with longer survival in glioblastoma patients receiving alkylating agents. Note that the median survival of patients with MGMT hypermethylation in the group 1 was 285 days (IQR = 323.75), and in the group without hypermethylation was significantly more than 338.5 days (IQR = 476) (p = 0.045). A similar trend was observed in the second group: in patients with hypermethylated MGMT, the median survival was 271 days (IQR = 337), the result is different from patients without hypermethylated MGMT – 342 days (IQR = 493) (p = 0.039). Conclusions: As a result, the application of the stereotactic approach of radiation therapy in the complex treatment of patients with glioblastoma and their classification by the MGMT methylation status with a cut-off point of 10% has the basis for improving the effectiveness and tolerability of radiation therapy and contributes to the overall survival of patients.

DNA methylation status of P2 promoter of IGF2 gene in growth restricted neonates

ObjectiveSmall for gestational age (SGA) is associated with epigenetic changes in DNA methylation status of various gene promoters as studied in cord blood of neonates and placenta samples of their mothers. Changes in methylation status of IGF-2 gene promoter may down regulate its expression and thereby control the weight and growth of babies. Objective of the current study was to determine the P2 promoter methylation status of IGF-2 gene in SGA neonates. Study designTwenty-eight growth restricted and 29 appropriate for gestational age (AGA) neonates were recruited in this study and umbilical cord blood samples were collected at birth. DNA samples were isolated and subjected to bisulfite modification and using specific primers methylation specific (MS) PCR was performed. The status of methylation in P2 promoter of IGF-2 was recorded after agarose gel electrophoresis. ResultsHypo methylation of P2 promoter of IGF-2 promoter was found in 64.3% of cord blood samples of growth restricted neonates as compared to 14% of AGA (p < 0.001). However, there was no correlation between methylation status and other risk factors of SGA. ConclusionIGF-2 P2 promoter hypo methylation is found in majority of cord blood samples of Indian growth restricted neonates.

The Impact of DNA Methylation on IL6 mRNA Levels in Hematinic Deficiency and Atopy-Associated Recurrent Aphthous Stomatitis Patients.

Objective To investigate the DNA methylation using pyrosequencing and its effects on the upregulation of IL6 mRNA in patients with recurrent aphthous stomatitis (RAS) in connection with hematinic deficiency and atopy. Material and Methods. This cross-sectional study was conducted at Dr. Hasan Sadikin Hospital, Bandung, from January–March 2019 and was approved by the Health Research Ethics Committee of Universitas Padjadjaran (Ethics No. 990/UN6.KEP/EC/2018). Furthermore, the subjects had RAS ulcers with a history of at least twice a year along with atopy and dietary imbalance with no history of recurrent intraoral herpes or any systemic diseases. This study was performed on 23 RAS patients and 21 healthy subjects, and the sampling was carried out consecutively. The blood samples were collected from all the subjects, and then, the DNA and RNA were extracted from the peripheral blood mononuclear cells (PBMCs). Consequently, the bisulfite-modified DNA was used to confirm the methylation status of the IL6 gene promoter through the pyrosequencing method. The methylation levels of the IL6 promoter were assessed by a reverse transcriptase-polymerase chain reaction technique. The gene expression of RAS and the control group was analyzed by the 2−ΔΔCT method. The statistical analysis using the Mann–Whitney U test was conducted to evaluate IL6 mRNA levels and DNA methylation with p value <0.05 considered to be statistically significant. Result The IL6 mRNA levels were approximately 1.88-fold in RAS patients, and there was a significant relationship between the expression of the IL6 gene and the increased risk of RAS (p < 0.001). It was reported that four out of six sites in the cytosine phosphate guanine (CpG) island IL6 promoter had a lower degree of methylation, and two other sites in patients with RAS had greater methylation compared with control, but not statistically significant. Conclusion This study showed the upregulation of IL6 mRNA levels in RAS patients compared to control. DNA methylation in the present study is at sites 566–658, whereas the location of the IL6 promoter is at sites 1–1684. Thus, it would be necessary conducting some research at other CpG sites of IL6 promoter islands to determine the status of DNA methylation.

Effect of DR4 Demethylation to the Proliferation and Apoptosis of Myeloid Leukemia K562 Cells

To investigate the effect of tumor necrosis factor death receptor (DR) 4 demethylation to the proliferation and apoptosis of myeloid leukemia K562 cells. The logarithmic phase of K562 cells were treated by desitabine (DCA) at 0, 0.8, 1.6 and 3.2 μmol/L, and the cells were divided into control group, DCA low dose group, DCA medium dose group and DCA high dose group respectively. The cells in control group were treated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 0.5 μg/ml for 24 h, and the cells were divided into TRAIL group. The cells in DCA high dose group were treated by TRAIL 0.5 μg/ml for 24 h, and were divided into DCA high dose + TRAIL group. Methylation-specific polymerase chain reaction (MS-PCR) was used to measure the methylation status of the DR4 gene promoter in the control group and DCA low, medium and high dose groups. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to determine the relative expression of DR4 mRNA and protein in the control group and DCA low, medium and high dose groups. Dime- thylthiazole (MTT) method was used to determine the inhibition rate of cell proliferation of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. Flow cytometry was used to determine the apoptotic rate of the cells in control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group. The cells in the control group were methylation-positive, the brightness of the methylation bands of the cells in the DCA low, medium, and high dose groups was gradually decreased to disappear, and the DCA high dose group showed negative for methylation. The relative expression of DR4 mRNA and protein in the control group, DCA low, medium and high dose groups was increased sequentially (r=0.624, 0.704). The inhibition rate of cell proliferation of the cells in the control group, DCA high dose group, TRAIL group, DCA high dose + TRAIL group was increased sequentially (r=0.653, 0.754, 0.709, 0.725) at 24, 48 and 72 h. DCA can reverse the methylation level of DR4 gene promoter in ML K562 cells and up-regulate the expression of DR4, which may enhance the proliferation inhibition and apoptosis promotion effects of TRAIL on K562 cells.

Methylation Status of Arabidopsis DNA Repair Gene Promoters During Agrobacterium Infection Reveals Epigenetic Changes in Three Generations

Agrobacterium tumefaciens is a unique pathogen with the ability to transfer a portion of its DNA, the T-DNA, to other organisms. The role of DNA repair genes in Agrobacterium transformation remains controversial. In order to understand if the host DNA repair response and dynamics was specific to bacterial factors such as Vir proteins, T-DNA, and oncogenes, we profiled the expression and promoter methylation of various DNA repair genes. These genes belonged to nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. We infected Arabidopsis plants with different Agrobacterium strains that lacked one or more of the above components so that the influence of the respective factors could be analysed. Our results revealed that the expression and promoter methylation of most DNA repair genes was affected by Agrobacterium, and it was specific to Vir proteins, T-DNA, oncogenes, or the mere presence of bacteria. In order to determine if Agrobacterium induced any transgenerational epigenetic effect on the DNA repair gene promoters, we studied the promoter methylation in two subsequent generations of the infected plants. Promoters of at least three genes, CEN2, RAD51, and LIG4 exhibited transgenerational memory in response to different bacterial factors. We believe that this is the first report of Agrobacterium-induced transgenerational epigenetic memory of DNA repair genes in plants. In addition, we show that Agrobacterium induces short-lived DNA strand breaks in Arabidopsis cells, irrespective of the presence or absence of virulence genes and T-DNA.

Evaluation of Methylation Profiles of An Epidermal Growth Factor Receptor Gene in a Head and Neck Squamous Cell Carcinoma Patient Group.

Upregulation of the epidermal growth factor receptor (EGFR) gene has shown an important impact on the development of head and neck cancers due to its important regulation role on multiple cell signaling pathways. The aim of this study was to investigate the methylation pattern of the promoter region of the EGFR gene between head and neck squamous cell carcinoma (HNSCC) patients and a control group. Forty-seven unrelated HNSCC patients, clinically diagnosed at the Department of Otorhinolaryngology, Dışkapı Yıldırım Beyazıt Training and Research Hospital, Ankara, Turkey, and 48 unrelated healthy volunteers from different geographic regions of Turkey, were included in this study. Methylation status of the promoter region of the EGFR gene was detected by methylation-specific-polymerase chain reaction (MS-PCR). The correlation between EGFR gene promoter methylation profiles and clinical characteristics were examined using the χ2 test. Methylation was observed in 79.0% of HNSCC patients, whereas this ratio was 90.0% in healthy individuals. The results show that promoter region methylation of the EGFR gene was not associated with HNSCC development in the studied Turkish patient group. In addition, the methylation status of the EGFR gene promoter was not found to be related to age, gender or tumor stage.

Glutathione S transferase T1 gene polymorphism and its promoter methylation are associated with breast cancer susceptibility in Egyptian women.

Breast cancer (BC) is one of the leading causes of cancer mortality in women. Glutathione S-transferase (GSTT1) is involved in activation of detoxification reactions and catalysis of chemicals conjugation with glutathione. GSTT1 genotype is a limiting factor for some environmental diseases. Epigenetic changes have an essential role in BC through inappropriate interaction between genomic and environmental risk factors. This study was directed to explore the association of BC risk with GSTT1 genetic variations and its methylation status in Egyptian women. This study included 100 healthy women as the control group and 100 patients were clinically and histologically diagnosed with breast cancer. All blood samples were used for genomic DNA extraction. GSTT1 genotyping was accomplished by multiplex PCR and methylation-specific PCR was used to analyze the GSTT1 promoter methylation status. Breast cancer patients showed significant incidence of null GSTT1 in relation to controls (p = 0.004). GSTT1 gene promoter methylation status showed significant difference between hypermethylated and unmethylated patients when compared with healthy subjects (p = 0.005). GSTT1 promoter methylation status was not significantly associated with null genotype. There was no significant association between GSTT1-null genotypes and BC stage in cases with or without family history, but for promotor methylation, there was significant association with stage III and IV breast cancer disease. GSTT1 null genetic variant and promoter hypermethylation in the GSTT region of the gene may be considered as critical risk factors for BC in Egyptian women.

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.

Association between MeCP2 and Smad7 in the pathogenesis and development of pathological scars

To explore the relationship between methylated binding protein 2 (MeCP2) and mothers against decapentaplegic homolog 7 (Smad7) in the pathogenesis and development of pathological scars. Immunohistochemistry, Western blot and real-time polymerase chain reaction (RT-PCR) were used to detect the expression of MeCP2 in different types of human scars and hypertrophic scars at different growth times. The methylation status of Smad7 gene promoter in different scar tissues was determined by methylation-specific PCR. After transfection with MeCP2-siRNA (small interfering RNA) in human keloid fibroblasts, MTT assay was used to assess the proliferation activity of keloid fibroblasts, while RT-PCR and Western blot assays were used to detect the expression levels of MeCP2, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), phospho-Smad2 (p-Smad2) and Smad7. MeCP2 was mainly expressed in the nucleus of fibroblasts. The mRNA and protein levels of MeCP2 were significantly higher in keloids than in hypertrophic scars, normal scars and normal skin (p<.05). The expression level of MeCP2 in hypertrophic scars during the growth period of <6 months was markedly higher than that of >6 months (p<.05). The methylation level of Smad7 was significantly higher in keloids compared to normal skin. After MeCP2 silencing, the proliferation rate of human keloid fibroblasts was decreased, the mRNA and protein levels of Smad7 were increased, and the expression levels of TGF-β1, α-SMA and p-Smad2 were decreased (p<.05). MeCP2 and Smad7 play an important role in formation of pathological scars. During keloid formation, MeCP2 weakens the inhibitory effect of Smad7 on p-Smad2/3 by downregulating the expression of Smad7, which in turn promotes fibrosis and scar hyperplasia.

ABCA1 gene promoter methylation and sudden cardiac death

Aim . A study of the association of the methylation of the promoter of the ABCA1 gene with sudden cardiac death (SCD). Materials and methods . The study design is based on the case-control principle. The SCD group included 150 men (mean age (46.7 ± 9.2) years) who died of sudden cardiac death according to forensic medical examination data (the main pathological diagnoses are acute circulatory failure, acute coronary insufficiency). The control group included 150 men (mean age (42.6 ± 1.2) years) who died suddenly, but not due to cardiovascular pathology. DNA was isolated by phenol-chloroform extraction from myocardial tissue. The methylation status of the ABCA1 gene promoter was assessed by methyl-specific polymerase chain reaction. The results obtained were statistically processed in SPSS 16.0 using Pearson’s test and Fisher’s test with Yates’ correction for continuity. P &lt; 0.05 was used as a level of significance. Results . Comparing the groups revealed statistically significant differences in the methylation status of the gene promoter ( p = 0.015). In the SCD group, the proportion of individuals whose ABCA1 gene promoter is methylated is statistically significantly higher compared to the control group ( p = 0.020; OR = 5.86; 95% CI (1.28–26.89)). Conclusion. Methylation of the promoter of the ABCA1 gene is associated with sudden cardiac death.

Methylation status of galectin-12 gene promoter in acute myeloid leukemia

Intro duction Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder, characterized by a block in differentiation of hematopoiesis, which results in the growth of a clonal population of neoplastic cells or blasts. In a previous study, the expression profiling of cancer-related galectins in AML showed upregulated expression of galectin-12 leading to cell cycle arrest. This observation pointed to the possibility that galectin-12 in AML could be a tumor-suppressor gene and a potential prognostic marker. Objective The aim was to study the methylation status of galectin-12 gene promoter in AML and its correlation with the expression of the gene and its potential effect on overall survival of patients. Patients and methods This study was carried out on 28 newly diagnosed AML cases. Expression of gal-12 gene was detected by reverse transcriptase-PCR, and the methylation status was examined by bisulfite genomic sequencing. Results Our results showed that 23/28 (82.1%) cases were negative for mRNA expression. The same group (82.1%) showed hypermethylated CpG island in the promoter region of LGALS12. Conclusion There is an association between LGALS12 expression and promoter hypermethylation [refers to the addition of a methyl group (CH3) covalently to the base cytosine (C) in the dinucleotide 5[Combining Acute Accent]-CpG-3[Combining Acute Accent]]. The mean survival of patients with unmethylated LGALS12 gene is significantly higher than methylated gene.

DNA methylation integratedly modulates the expression of Pit-Oct-Unt transcription factors in esophageal squamous cell carcinoma.

Background: Dysregulation of Pit-Oct-Unc family transcription factors has been implicated in esophageal squamous cell carcinoma (ESCC). In this study, we evaluated the expression and promoter methylation status of Octamer (OCT) transcription factor genes in human ESCC clinical specimens to investigate the mechanism underlying this observation along with the clinical significance.Methods: Total DNA or RNA was extracted from ESCC tissue specimens and the mRNA level of genes encoding the transcription factors OCT1, OCT2, OCT3/OCT4, OCT5, OCT7, OCT9, and OCT11 were evaluated by quantitative PCR. The DNA methylation status of gene promoters was assessed by bisulfite pyrosequencing and next-generation sequencing. The relationship between the expression of these transcription factors and ESCC proliferation was investigated in vitro and in vivo with the colony formation assay and a mouse xenograft tumor model, respectively. We also examined the correlation between OCT gene expression and promoter methylation and clinicopathologic characteristics of ESCC.Results: OCT1 was upregulated whereas OCT4, OCT6, and OCT11 were downregulated in ESCC compared to non-tumor tissue. OCT2, OCT7, and OCT9 were undetected in all samples. OCT1, OCT6, and OCT11 levels were negatively correlated with the methylation of their respective promoters, but there was no relationship between OCT4 expression and promoter methylation status.Conclusion: Changes in promoter methylation rate underlie the observed alterations in OCT1, OCT6, and OCT11 expression in ESCC, whereas another mechanism is likely responsible for the dysregulation of OCT4.

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

Characterization of primary glioma cell lines derived from the patients according to 2016 CNS tumour WHO classification and comparison with their parental tumours.

Gliomas represent about 80% of primary brain tumours and about 30% of malignant ones, which today don't have a resolution therapy because of their variability. A valid model for the study of new personalized therapies can be represented by primary cultures from patient's tumour biopsies. In this study we consider 12 novel cell lines established from patients' gliomas and immunohistochemically and molecularly characterized according to the newly updated 2016 CNS Tumour WHO classification. Eight of these lines were glioblastoma cells, two grade III glioma cells (anaplastic astrocytoma and oligo astrocytoma) and two low grade glioma cells (grade II astrocytoma and oligodendroglioma). All cell lines were analysed by immunohistochemistry for specific glioma markers, respectively VIMENTIN, GFAP, IDH1R132, and ATRX. The methylation status of the MGMT gene promoter was also determined in all lines. The comparison of the immunohistochemical characteristics and of the MGMT methylation status of the lines with the tissues of origin shows that the cells in culture maintain the same characteristics. Human cancer cell lines represent a support in the knowledge of tumour biology and in drug discovery through its facile experimental manipulation. NCT04180046.