Methylation-sensitive High-resolution Melting Research Articles

A case control study investigating the methylation levels of GHRL and GHSR genes in alcohol use disorder.

Alcohol use disorder (AUD) is a relapsing disease described as excessive use of alcohol. Evidence of the role of DNA methylation in addiction is accumulating. Ghrelin is an important peptide known as appetite hormone and its role in addictive behavior has been identified. Here we aimed to determine the methylation levels of two crucial genes (GHRL and GHSR) in ghrelin signaling and further investigate the association between methylation ratios and plasma ghrelin levels. Individuals diagnosed with (n = 71) and without (n = 82) AUD were recruited in this study. DNA methylation levels were measured through methylation-sensitive high-resolution melting (MS-HRM). Acylated ghrelin levels were detected by ELISA. The GHRL rs696217 polymorphism was analyzed by the standard PCR-RFLP method. GHRL was significantly hypermethylated (P < 0.0022) in AUD between 25 and 50% methylation than in control subjects but no significant changes of GHSR methylation were observed. Moreover, GHRL showed significant positive correlation of methylation ratio between 25 and 50% with age. A significant positive correlation between GHSR methylation and ghrelin levels in the AUD group was determined (P = 0.037). The level of GHRL methylation and the ghrelin levels showed a significant association in the control subjects (P = 0.042). GHSR and GHRL methylation levels did not change significantly between control and AUD groups. However, GHRL and GHSR methylations seemed to have associations with plasma ghrelin levels in two groups. This is the first study investigating the DNA methylation of GHRL and GHSR genes in AUD.

DNA methylation-altered genes in the rat hippocampal neurogenic niche after continuous exposure to amorphous curcumin

Rat offspring who are exposed to an amorphous formula of curcumin (CUR) from the embryonic stage have anti-anxiety-like behaviors, enhanced fear extinction learning, and increased synaptic plasticity in the hippocampal dentate gyrus (DG). In the present study, we investigated the links between genes with altered methylation status in the neurogenic niche and enhanced neural functions after CUR exposure. We conducted methylation and RNA sequencing analyses of the DG of CUR-exposed rat offspring on day 77 after delivery. Methylation status and transcript levels of candidate genes were validated using methylation-sensitive high-resolution melting and real-time reverse-transcription PCR, respectively. In the CUR group, we confirmed the hypermethylation and downregulation of Gpr150, Mmp23, Rprml, and Pcdh8 as well as the hypomethylation and upregulation of Ppm1j, Fam222a, and Opn3. Immunohistochemically, reprimo-like+ hilar cells and protocadherin-8+ granule cells were decreased and opsin-3+ hilar cells were increased by CUR exposure. Both reprimo-like and opsin-3 were partially expressed on subpopulations of glutamic acid decarboxylase 67+ γ-aminobutyric acid-ergic interneurons. Furthermore, the transcript levels of genes involved in protocadherin-8-mediated N-cadherin endocytosis were altered with CUR exposure; this was accompanied by Ctnnb1 and Syp upregulation and Mapk14, Map2k3, and Grip1 downregulation, suggesting that CUR-induced enhanced synaptic plasticity is associated with cell adhesion. Together, our results indicate that functionally different genes have altered methylation and expression in different neuronal populations of the hippocampal neurogenic niche, thus enhancing synaptic plasticity after CUR exposure.

L-carnitine fails to rescue chemotherapy injured ovaries by epigenetic changes of transcription factors

Cyclophosphamide (CP), as an anti-cancer drug, is frequently used to treat various types of cancer. A decreased number of ovarian follicles impaired normal ovarian function, and subsequent premature ovarian failure (POF) presented as a side effect of cyclophosphamide usage. These events may eventually affect the fertility rate of individuals. The present study showed the effect of cyclophosphamide on ovarian reserves and the protective effect of L-carnitine (LC) as an antioxidant to prevent POF.To design the study, six to eight-week-old NMRI female mice were divided into three groups: control, cyclophosphamide (CP), and cyclophosphamide + L-carnitine (CP + LC). Mice received drugs intraperitoneally (IP) for 21 days. In the following 24 h after the last injection, both ovaries were used to evaluate the expression of Sohlh1 and Lhx8 genes by Real-time PCR. Furthermore, the alteration of Lhx8 promoter methylation was examined by Methylation-sensitive high-resolution melting analysis (MS-HRM).The present data showed the negative effect of CP on regulator genes of oogenesis including Sohlh1 and Lhx8. In addition, an examination of the epigenetic status of the Lhx8 gene showed a change in promoter methylation of this gene following cyclophosphamide injection. Although, L-carnitine is an effective antioxidant in relieving oxidative stress caused by cyclophosphamide and its damage, in the present study, however, the use of L-carnitine failed to protect the ovaries from changes caused by CP injection. So, using cyclophosphamide can alter the expression of folliculogenesis genes through its effects on epigenetic changes and may cause POF. The results of the present study showed that L-carnitine consumption can’t protect the ovaries against the adverse effects of CP.

Age estimation of captive Asian elephants (Elephas maximus) based on DNA methylation: An exploratory analysis using methylation-sensitive high-resolution melting (MS-HRM)

Age is an important parameter for bettering the understanding of biodemographic trends—development, survival, reproduction and environmental effects—critical for conservation. However, current age estimation methods are challenging to apply to many species, and no standardised technique has been adopted yet. This study examined the potential use of methylation-sensitive high-resolution melting (MS-HRM), a labour-, time-, and cost-effective method to estimate chronological age from DNA methylation in Asian elephants (Elephas maximus). The objective of this study was to investigate the accuracy and validation of MS-HRM use for age determination in long-lived species, such as Asian elephants. The average lifespan of Asian elephants is between 50–70 years but some have been known to survive for more than 80 years. DNA was extracted from 53 blood samples of captive Asian elephants across 11 zoos in Japan, with known ages ranging from a few months to 65 years. Methylation rates of two candidate age-related epigenetic genes, RALYL and TET2, were significantly correlated with chronological age. Finally, we established a linear, unisex age estimation model with a mean absolute error (MAE) of 7.36 years. This exploratory study suggests an avenue to further explore MS-HRM as an alternative method to estimate the chronological age of Asian elephants.

Non-invasive age estimation based on faecal DNA using methylation-sensitive high-resolution melting for Indo-Pacific bottlenose dolphins.

Age is necessary information for the study of life history of wild animals. A general method to estimate the age of odontocetes is counting dental growth layer groups (GLGs). However, this method is highly invasive as it requires the capture and handling of individuals to collect their teeth. Recently, the development of DNA-based age estimation methods has been actively studied as an alternative to such invasive methods, of which many have relied on used biopsy samples. However, if DNA-based age estimation can be developed from faecal samples, age estimation can be performed entirely non-invasively. We developed an age estimation model using the methylation rate of two gene regions, GRIA2 and CDKN2A, measured through methylation-sensitive high-resolution melting (MS-HRM) from faecal samples of wild Indo-Pacific bottlenose dolphins (Tursiops aduncus). The age of individuals was known through conducting longitudinal individual identification surveys underwater. Methylation rates were quantified from 36 samples collected from 30 individuals. Both gene regions showed a significant correlation between age and methylation rate. The age estimation model was constructed based on the methylation rates of both genes which achieved sufficient accuracy (after LOOCV: MAE = 5.08, R2 = 0.33) for the ecological studies of the Indo-Pacific bottlenose dolphins, with a lifespan of 40-50 years. This is the first study to report the use of non-invasive faecal samples to estimate the age of marine mammals.

Determination of the Relationship Between DNA Methylation Status of KLOTHO and ARNTL Genes With Hypertension.

Hypertension is a multifactorial chronic disease due to the interaction of environmental factors with genetic alteration. KLOTHO and ARNTL genes play an important role in the development of hypertension. Therefore, we analyzed the methylation status of KLOTHO and ARNTL genes by using methylation-sensitive high-resolution melting (MSHRM) in a total of 78 hypertensive and 49 control subjects. In this study, we could not identify a significant association between KLOTHO and ARNTL methylation and the hypertensive phenotype. Moreover, we could not find a direct association between KLOTHO and ARNTL methylation and the fasting blood sugar, triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, sodium (Na), creatinine (Cr), potassium (K), and urea levels in hypertensive patients. However, we found a significant difference between the methylated KLOTHO hypertensive patients and the unmethylated KLOTHO control subjects for potassium (K).

Association of Promoter Methylation Patterns with Expression of MAPK14 in Tissue of Papillary Thyroid Cancer Patients.

Thyroid cancer is one of the most prevalent malignancies worldwide. Genetic and epigenetic alterations are one of the main causes of thyroid tumor that is responsible to the activation of oncogenes as well as the inactivation of tumor suppressor genes. This research aimed to investigate the relationship of promoter methylation patterns with the expression of P38α in Iranian patients with thyroid cancer. We collected 40 thyroid tumor samples and 40 adjacent normal thyroid samples from 40 Iranian patients with papillary thyroid cancer. The promoter methylation pattern of P38α gene was investigated by methylation-sensitive high-resolution melting (MS-HRM) method. Moreover, mRNA expression of P38α was investigated by Real-Time PCR method. Further validation of the obtained results was performed by the Cancer Genome Atlas (TCGA) dataset. The obtained results indicated that the expression of the P38α (MAPK-14) gene in the thyroid cancer sample was considerably higher than tumor margin sample. Also, P38α gene promoter methylation was higher in thyroid margin tissue as compared to tumor tissue. These results were additionally confirmed by TCGA analysis. The receiver operating characteristic (ROC) curve analysis showed a high accuracy of P38α gene expression as a diagnostic biomarker for thyroid malignancy. Our study demonstrated that the P38α expression level gene was associated with thyroid cancer pathogenesis among the Iranian population. We suggested that this gene expression might be used as a biomarker for diagnosis of thyroid tumor.

Institutional Validation Study Inferring 2% MGMT Methylation Positive Impact on Survival in Newly Diagnosed Glioblastoma (GBM) Patients

O6-methylguanine DNA methyltransferase (MGMT) MATERIALS/METHODS: is a well-established prognostic factor in patients with newly diagnosed glioblastoma (GBM). However, there is no consensus on a standardized method of threshold for MGMT testing. Previous studies have reported levels of as little as 1-3% to confer better prognosis. This study reports a single institutional experience of determining methylation status via methylation-sensitive high-resolution melting (MS-HRM). Previous literature suggests 10% cutoff for MGMT methylation using MS-HRM. We hereby report clinical outcomes using a lower threshold of 2%. GBM patients treated at our institution retrospectively reviewed between the years 2013 and 2022 were included in the study. Patients who received hypofractionated radiation (<60 Gy) were excluded. All patients had MS-HRM test for MGMT methylation status. A real-time PCR assay was used to amplify a 62 base-pair region of MGMT for both methylated and unmethylated alleles. PCR products underwent HRM analysis and the fraction of methylated DNA was determined by comparison with a standard curve. Clinical data were collected retrospectively. Kaplan-Meier and log-rank tests were performed to compare survival. A total of 181 patients with newly diagnosed GBM were initially included in this study. 42 patients treated with hypofractionated radiation were excluded. All patients received concurrent and maintenance temozolomide. Median age was 61.5 years. A total of 84 patients had MGMT methylation levels <2%, and 55 patients had MGMT methylation level ≥ 2% with a median methylation level of 28.5% (Range 0.8%-100%). Patients with MGMT methylation level ≥ 2% had an improved median overall survival (25.1 vs 16.0 months; p = 0.006) and improved median progression free survival (11.3 vs 7.9 months; p = 0.017). In a multivariable mode that included age, use of tumor-treating fields, KPS, sex, and BMI, only age, KPS, and MGMT remained significant. Our institutional review confirmed low level of MGMT hypermethylation (≥ 2%) predicts improved outcome in patients with newly diagnosed GBM. Further investigation on optimal cut off level for MGMT methylation is still warranted.

METHYLATION PATTERN OF THE RUNX3 GENE AND ITS IMPACT ON RESPONSE TO IMATINIB THERAPY IN CHRONIC MYELOID LEUKEMIA

BackgroundChronic Myelogenous Leukemia (CML) is a clonal stem cell disease with deregulated tyrosine kinase activity of BCR-ABL. DNA methylation in gene promoters influences disease development, therapeutic response, and clinical outcome in various diseases.&#x0D; ObjectivesThis study aimed to investigate the methylation pattern in promoter regions of the runt-related transcription factor 3 (RUNX3) gene and its effect on the response to imatinib therapy, among CML patients.&#x0D; Patients and MethodsBlood samples were collected from 100 participants from November 2020 to February 2022, in two groups 75 samples of CML patients and 25 samples of healthy controls, genomic DNA was extracted from the samples by using the salting-out method, then Isolated DNA was treated with sodium bisulfite. By Methylation-Sensitive High-Resolution Melting (MS-HRM), the samples were analyzed.&#x0D; ResultsThe promoter sequence methylation level is significantly higher (P&lt;0.05) in DNA samples from CML patients than in the control group, the methylation level in most of the healthy control samples 96% was detected between 0-24% while in CML patients was 16%, in 25-49%, RUNX3 methylation levels in Health individuals were 4%, moreover in CML patients were 24% also, in 50-100% of methylation level, health control was 0%, in CML patient showed 60%. According to methylation detection of RUNX3 among Imatinib resistance and good response, the results of IM resistance were detected between 0-40% was 15.21%, Furthermore, in good response was 79.31%. In other hand at 41-100% RUNX3 methylation levels in IM resistance was 84.78%, while a good response was recorded at 20.68%.&#x0D; ConclusionThe study concluded that RUNX3 gene hypermethylation often occurs in CML patients. Additionally, there is a strong correlation between RUNX3 hypermethylation and poor imatinib respondents. We found that both IM resistance and CML patients have higher levels of RUNX3 gene hypermethylation. Moreover, research on a larger sample size is required to validate these findings, which may serve as possible indicators of disease progression and imatinib resistance.

Contribution of constitutional BRCA1 promoter methylation to early-onset and familial breast cancer patients from Pakistan.

Constitutional BRCA1 promoter methylation has been identified as a potential risk factor for breast cancer (BC) in the Caucasian population. However, this data is lacking for BC patients of Asian origin. Therefore, we assessed the contribution of constitutional BRCA1 promoter methylation in Pakistani BC patients. A total of 385 BRCA1/2-negative index BC patients (197 early-onset BC (≤ 30years), 152 familial BC, 17 familial BC and ovarian cancer, 19 male BC) and 107 healthy controls were screened for the constitutional BRCA1 promoter methylation by methylation-sensitive high-resolution melting assay. Overall, 131 patients displayed triple-negative BC (TNBC) and 254 non-TNBC phenotypes. The prevalence of BRCA1 promoter methylation was calculated based on clinicopathological characteristics using univariable and multivariable logistic regression models. Constitutional BRCA1 promoter methylation was identified in 19.5% (75/385) of BC patients and 13.1% (14/107) of controls. The frequency of methylation was higher in early-onset BC (23.4% vs. 13.1%, P = 0.035) and TNBC patients (29.0% vs. 13.1%, P = 0.004) compared to controls. Methylation was also more prevalent in patients with high-grade than low-grade tumors (21.7% vs. 12.2%, P = 0.034) and progesterone receptor (PR)-negative than PR-positive tumors (26.0% vs. 13.9%, P = 0.004). Constitutional BRCA1 promoter methylation remained independently associated with TNBC phenotype (odds ratio 1.99; 95% CI 1.12-3.54; P = 0.02) after adjusting for BC diagnosis age, tumor grade, ER, and PR status. Constitutional BRCA1 promoter methylation is associated with TNBC and can serve as a non-invasive blood-based biomarker for Pakistani TNBC patients.

Olaparib in HR-deficient (HRD), metastatic triple-negative breast cancer (TNBC) and relapsed ovarian cancer (ROC) without germline mutations in BRCA1 or BRCA2: Phase 2 EMBRACE trial.

e15079 Background: Homologous recombination deficiency (HRD) caused by germline BRCA1/2 mutations (gBRCAm) sensitize high grade serous ovarian cancers (HGSOC) and TNBC to PARP inhibitors (PARPi) such as olaparib. Loss of function of BRCA1 or RAD51C, due to promoter methylation or mutation of non-BRCA HR genes may also confer sensitivity to PARPi. This investigator-initiated, proof-of-concept trial evaluated olaparib in platinum(Pt)-sensitive, relapsed HGSOC and metastatic TNBC, with HRD due to mechanisms other than gBRCAm. EMBRACE is the first trial evaluating PARPi in loss of function of BRCA1/ RAD51C by promoter methylation. Methods: Single-arm, phase 2 trial of olaparib 300 mg orally twice daily in adults with metastatic TNBC or Pt-sensitive relapsed HGSOC and HRD due to mechanisms other than gBRCAm. 1 line of prior Pt-chemotherapy was allowed (HGSOC); additional lines of non-Pt therapy were allowed (TNBC). Tumor BRCA1/RAD51C promoter methylation was determined by methylation-sensitive high-resolution melting PCR and next generation sequencing (NGS, Pillar Biosciences BRCA1 &amp; RAD51C Methylation test, high methylation threshold 70%). Unmethylated cases underwent NGS HR gene mutation testing (Pillar Biosciences HRD test, paired tumor/normal). The primary outcome was objective tumor response rate (OTRR) at 6 months(m). Secondary objectives: progression-free survival (PFS), OTRR according to HR gene mutation type, safety. Tertiary: associations between biomarkers and clinical outcomes. Results: 22 participants (15 HGSOC, 7 TNBC) enrolled from 208 screened (Sep 2018 - Mar 2022). Methylation was detected in 8/15 HGSOC (all BRCA1) and 5/7 TNBC (4 BRCA1, 1 RAD51C). Both cohorts included mutations in gBRIP1 (1), gPALB2 (1), gRAD51C (3), sBRCA1 (2), with 1 COSMIC mutational signature 3, and 1 positive HRD score in HGSOC. OTRR at 6m was 40% in HGSOC (6/15: 5 partial, 1 complete), and 0% in TNBC (0/7). OTRR was 38% (3/8) in methylated tumors vs 43% (4/7) with other HRD. Stable disease at 6m was 47% (7) in HGSOC and 43% (3) in TNBC durations were 7m in HGSOC and 3m in TNBC. PFS was 53% at 6m and 25% at 12m in HGSOC, vs 17% at 6m and NE at 12m in TNBC. TNBC 2-3 prior lines of therapy. Conclusions: Olaparib demonstrated clinically relevant activity in HGSOC without gBRCAm. Objective responses were seen in HGSOC with methylated BRCA1. Olaparib had limited activity in heavily pre-treated TNBC. Further research is needed into the effects of non-Pt chemotherapy on methylation of BRCA1/RAD51C to improve selection of HGSOC and TNBC for PARPi. (ANZCTRN12617000855325) Acknowledgements: Funding from Cancer Australia and the NBCF(PS-15-048), provision of study drug plus an untied educational grant from AstraZeneca and its group of companies. EMBRACE was led by the NHMRC CTC, University of Sydney in collaboration with ANZGOG, BCT-ANZ, and GCCTI. Clinical trial information: ACTRN12617000855325 .

Abstract 4723: DNA G-quadruplex facilitates MYC enhancer-promotor interaction

Abstract Background: G-quadruplexes (G4) are nucleic acid secondary structures formed in guanine-rich regions of DNA or RNA. The biological significance of G4 has been attributed to the regulation of gene expression, and more recently, to the stabilization and generation of enhancer-promotor interaction. Since G4 are enriched in the promoter region of the proto-oncogene MYC, we sought to investigate the role of G4 in stabilizing the enhancer-promoter loop using the locus 8q24 (hg38) 127,700,000-128,500,000 that contains the MYC gene as a model. Methods: Two different cell lines were used, the spontaneously immortalized, non-oncogenic human epidermal keratinocyte (HaCaT), in which the enrichment of G4 in the MYC promoter was demonstrated, and the primary normal human epidermal keratinocyte (nHEK) that is depleted of G4 in MYC promoter. Both cell lines were exposed to the G4 selective stabilizer 360-A drug and the half-maximal inhibitory concentration (IC50) could be calculated. Gene expression was accessed by RT-qPCR and DNA methylation by Methylation-Sensitive High Resolution Melting (MS-HRM). Chromatin Conformation Capture (3C-qPCR) technique was performed interrogating the MYC gene (chr8:127,733,798-127,744,729) and the putative enhancer in the lncRNA PVT1 gene (chr8:127,914,509-127,922,393) to compare the frequency of promoter-enhancer interaction between the two cell lines. Results: The HaCaT cells showed interaction between the MYC promoter and the distal region. The nHEK cells that are not enriched for G4 showed no promoter interaction with the distal region. The use of 360-A in IC50 did not change the results. The gene expression analysis of the MYC showed higher expression in HaCaT cells compared to nHEK. There was no difference in DNA methylation in the MYC promoter between the two cell lines. Conclusions: The data suggest an important role of G4 for the interaction of the MYC promoter and downstream enhancer. Additional analyzes of other genomic regions enriched for G4 should be performed to confirm the mechanism. Citation Format: Dieila Giomo De Lima, Gustavo Narvaes Guimarães, Bianca Caroline Bianco, Ana Paula De Souza, Aline Cristiane Planello. DNA G-quadruplex facilitates MYC enhancer-promotor interaction. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4723.

PTEN promoter methylation and expression in endometrial cancer tissues.

Introduction: Some gene expression regulation in cancers can be controlled by epigenetic change like methylation. PTENpromoter methylation and expression were evaluatedin endometrial cancer. Methods: The study was run on 39 tumor tissues of endometrial cancer patients and 41 normal endometrial tissues. After total RNA extraction, cDNAsynthesis was done by reverse transcription of the total (real-time PCR) using SYBER Green master mix. DNA extraction and bisulfite treatment were conducted and methylation was semiquantified by the methylation-sensitive high-resolution melting method. Finally, promoter methylation quantification of the total number of 25 tumors and 22 non-neoplastic tissues was done. Results: PTEN gene expression showed a significant decrease in endometrial cancer tissues. Promoter methylation was significantly lower in the non-neoplastic group (7.2; p<0.001). In addition, PTEN promoter methylation was observed in 52.0% of tumor tissues compared with 13.6% in thenon-neoplastic group(p=0.06). There were no significant correlations between PTEN expression and methylation andclinicopathological features in endometrial cancer patients (p>0.05). Conclusion: PTEN gene expression in endometrial cancer tissues decreased because of its promoter hypermethylation.

Variability, Expression, and Methylation of IL-6 and IL-8 Genes in Bladder Cancer Pathophysiology.

Bladder cancer (BC) is the 10th most common form of cancer globally, but its complete aetiology is still unknown. Nevertheless, there is evidence that chronic inflammation plays a role in the development and progression of BC. Therefore, the presented study aimed to detect a potential association between selected single nucleotide polymorphisms (SNPs)-rs1800797 and rs2069845 in IL-6 and rs2227307 in IL-8-and BC development, as well as to identify the impact of BC on the level of expression and methylation of IL-6 and IL-8 promoters in PBMCs with the use of the TaqMan SNP genotyping assay, TaqMan gene expression assay, and methylation-sensitive high-resolution melting techniques. We did not find any association between the genotypes and combined genotypes of all studied polymorphisms and the occurrence of BC. However, we found that BC patients were characterised by decreased IL-6 and IL-8 mRNA expression levels compared to the controls. Additionally, the methylation status of the IL-6 promoter was higher in controls than in BC patients. Our findings suggest that inflammation may be involved in the development and progression of BC.

Joint Effects Between CDKN2B/P15 Methylation and Environmental Factors on the Susceptibility to Gastric Cancer.

The incidence of gastric cancer has long been at a high level in China, seriously affecting the health of Chinese people. This case‒control study was performed to identify gene methylation biomarkers of gastric cancer susceptibility. A total of 393 gastric cancer cases and 397 controls were included in this study. Gene methylation in peripheral blood leukocytes was detected by a methylation-sensitive high-resolution melting method, and the Helicobacter pylori antibody presence was semi-quantified in serum by ELISA. Individuals with total methylation of CDKN2B/P15 had a 1.883-fold (95%CI: 1.166-3.040, P = 0.010) risk of gastric cancer compared with unmethylated individuals. Individuals with both CDKN2B/P15 and NEUROG1 methylation had a higher risk of gastric cancer (OR = 2.147, 95% CI: 1.137-4.073, P = 0.019). The interaction between CDKN2B/P15 and NEUROG1 total methylation on gastric cancer risk was affected by the pattern of adjustment. In addition, the joint effects between CDKN2B/P15 total methylation and environmental factors, such as freshwater fish intake (OR = 6.403, 95% CI = 2.970-13.802, P < 0.001), irregular diet (OR = 5.186, 95% CI = 2.559-10.510, P < 0.001), unsanitary water intake (OR = 2.238, 95% CI = 1.144-4.378, P = 0.019), smoking (OR = 2.421, 95% CI = 1.456-4.026, P = 0.001), alcohol consumption(OR = 2.163, 95% CI = 1.309-3.576, P = 0.003), and garlic intake(OR = 0.373, 95% CI = 0.196-0.709, P = 0.003) on GC risk were observed, respectively. However, CDKN2B/P15 and NEUROG1 total methylation were not associated with gastric cancer prognosis. CDKN2B/P15 methylation in peripheral blood may be a potential biomarker for evaluating susceptibility to gastric cancer. The joint effects between CDKN2B/P15 methylation and environmental factors may also contribute to gastric cancer susceptibility.

Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile-The Example of SRXN1 Gene.

The role of catechins in the epigenetic regulation of gene expression has been widely studied; however, if and how this phenomenon relates to the redox properties of these polyphenols remains unknown. Our earlier study demonstrated that exposure of the human colon adenocarcinoma HT29 cell line to these antioxidants affects the expression of redox-related genes. In particular, treatment with (-)-epigallocatechin (EGC) downregulated transcription of gene encoding sulfiredoxin-1 (SRXN1), the peroxidase involved in the protection of cells against hydrogen peroxide-induced oxidative stress. The aim of this study was to investigate whether the observed SRXN1 downregulation was accompanied by changes in the DNA methylation level of its promoter and, if so, whether it was correlated with the redox properties of catechins. The impact on DNA methylation profile in HT29 cells treated with different concentrations of five catechins, varying in chemical structures and standard reduction potentials as well as susceptibility to oxidation, was monitored by a methylation-sensitive high-resolution melting technique employing the SRXN1 promoter region as a model target. We demonstrated that catechins, indeed, are able to modulate DNA methylation of the SRXN1 gene in a redox-related manner. The nonlinear method in the statistical analysis made it possible to fish out two parameters (charge transfer in oxidation process Qox and time of electron transfer t), whose strong interactions correlated with observed modulation of DNA methylation by catechins. Based on these findings, we present a proof-of-concept that DNA methylation, which limits SRXN1 expression and thus restricts the multidirectional antioxidant action of SRXN1, may represent a mechanism protecting cells against reductive stress caused by particularly fast-reacting reductants such as EGC and (-)-epicatechin gallate (ECG) in our study.

Association between the methylation of CpG islands in JAK-STAT pathway-related genes and colorectal cancer

BackgroundAberrant promoter methylation of CpG islands plays an important role in carcinogenesis. However, the association between the DNA methylation of JAK-STAT pathway-related genes in peripheral blood leukocytes and colorectal cancer (CRC) susceptibility remains unclear. MethodsWe conducted a case-control study of 403 patients with CRC and 419 cancer free controls, and the DNA methylation levels of JAK2, STAT1, STAT3, and SOCS3 in peripheral blood samples from all subjects were assessed using a methylation-sensitive high-resolution melting (MS-HRM) analysis. ResultsCompared with controls, the methylation of the JAK2, STAT1 and SOCS3 genes increased the CRC risk (ORadjusted=1.96, 95% CI, 1.12–3.41, P=0.01; ORadjusted=5.37, 95% CI, 3.74–7.71, P<0.01; ORadjusted=3.30, 95% CI, 1.58–6.87, P<0.01). In the multiple CpG site methylation (MCSM) analysis, a high MCSM value denoted an increased CRC risk (ORadjusted=4.97, 95% CI, 3.34–7.37, P<0.01). ConclusionIn peripheral blood, the methylation of JAK2, STAT1, and high levels of MCSM are promising biomarkers for CRC risk.

Methylation of the tumor associated genes in head and neck squamous cell carcinoma

Introduction. Head and neck cancer is a heterogenous group of malignant tumors of different etiologies, molecular mechanisms of which are still insufficiently studied.Aim. Investigation of DNA methylation status of some tumor associated genes (RASSF1A, RASSF2, RASSF5, CDO1, MEST and WIF1) in patients with head and neck squamous cell carcinoma.Materials and methods. The DNA methylation level of normal and tumor tissues was analyzed using bisulfite conversion and methylation-sensitive high-resolution melting in 25 patients (21 men and 4 women) diagnosed with neck squamous cell carcinoma.Results. There were significant differences in levels of DNA methylation between tumor and normal tissues in the CDO1 and WIF1 genes in all groups and subgroups of patients (larynx and other cancers, squamous cell carcinoma keratinizing and non-keratinizing, primary and recurrent tumor, smokers and non-smokers). The methylation level in the CDO1 gene in tumor tissue was significantly increased in the T4 and T3 stage subgroups compared to T2.Conclusion. The increased level of methylation of the CDO1 and WIF1 genes, as well as changes in their expression are among the molecular mechanisms involved in the neck squamous cell carcinoma development. They can be considered as prognostic and diagnostic markers for this pathology.

Promoter methylation levels of RASSF1 and ATIC genes are associated with lung cancer in Iranian patients.

Epigenetic alterations like methylation of tumor suppressor genes or oncogenes, in respiratory epithelium have been associated with lung cancer. Hypermethylation of genes promoter is an epigenetic event, and is responsible to tumor suppressor genes inactivation as well as oncogenes activation. This study aimed to assess the role of methylation status in promoter of RASSF1 and ATIC genes their potential implication in the pathogenesis of lung tumor in Iranian patients. In this study, we collected 100 tissue samples (50lung cancer tissues and 50 adjacent non-cancerous lung tissues) from Iranian lung cancer patients. The genomic DNA was extracted, and methylation status of both RASSF1 andATIC genes was investigated by methylation-sensitive high-resolution melting (MS-HRM) assay technique and Real-Time PCR. Cancer Genome Atlas (TCGA) dataset wasalso analyzed for further validation of the gene's methylation. Methylation of RASSF1 gene promoter was significantly higher in lung tumor tissues. However, promoter methylation levels of ATIC gene was significantly lower in lung tumor tissues. These results were additionally confirmed by TCGA analysis. Promoter methylation of both RASSF1 and ATIC genes was significantly associated with lymph node metastasis, and clinical stage of lung cancer. Thereceiver operating characteristic (ROC) curve analysis indicated a high accuracy of promoter methylation in these genes as a diagnostic biomarker for lung cancer. Methylation levels of both RASSF1 and ATIC genes promoters were associated with lung cancer pathogenesis in Iranian population, and may be a suitable biomarker for diagnosis and prognosis of lung cancer in early stage of tumorigenesis.

Methylation-sensitive high-resolution melting analysis of the USP44 promoter can detect early-stage hepatocellular carcinoma in blood samples

Hepatocellular carcinoma (HCC) is dangerous cancer that often evades early detection because it is asymptomatic and an effective detection method is lacking. For people with chronic liver inflammation who are at high risk of developing HCC, a sensitive detection method for HCC is needed. In a meta-analysis of The Cancer Genome Atlas pan-cancer methylation database, we identified a CpG island in the USP44 promoter that is methylated specifically in HCC. We developed methylation-sensitive high-resolution melting (MS-HRM) analysis to measure the methylation levels of the USP promoter in cell-free DNA isolated from patients. Our MS-HRM assay correctly identified 40% of patients with early-stage HCC, whereas the α-fetoprotein test, which is currently used to detect HCC, correctly identified only 25% of early-stage HCC patients. These results demonstrate that USP44 MS-HRM analysis is suitable for HCC surveillance. [BMB Reports 2022; 55(11): 553-558].