It remains unknown about molecular mechanisms underlying the transition of somatic cells into male germ cells. It is observed that RAD21L1 transcript is upregulated during reprogramming of Sertoli cells into human spermatogonial stem cells (SSCs) by overexpressing DAZ family genes. Significantly, RAD21L1 overexpression transits Sertoli cells into phenotypic and functional human SSCs with high safety. RNA sequencing shows that DNMT1 is expressed at a higher level by RAD21L1 overexpression when Sertoli cells are reprogrammed to become human SSCs. Whole genome bisulfite sequencing elucidates that RAD21L1 modulates DNA methylation to reprogram Sertoli cells into human SSCs, and RAD21L1 interacts with DNMT1 in human SSCs generated from Sertoli cells. Intriguingly, RAD21L1 mutation results in the decreases in stemness maintenance of human SSCs and DNMT1 expression levels. Notably, RAD21L1 mutations are positively related to risk of non-obstructive azoospermia (NOA) and male infertility. Collectively, these results implicate that RAD21L1 is sufficient and effective for reprogramming human Sertoli cells to SSCs through modulating DNMT1 and RAD21L1 mutations leads to NOA. This study is of particular significance because it provides a novel molecular mechanism that reprograms human somatic cells into human SSCs and it could offer invaluable gametes for treating male infertility.

The lack of quantitative methylation reference datasets (ground truth) and cross-laboratory reproducibility assessment hinders clinical translation of epigenome-wide sequencing technologies. Using certified Quartet DNA reference materials, here we generate 108 epigenome-sequencing datasets across three mainstream protocols (whole-genome bisulfite sequencing, enzymatic methyl-seq, and TET-assisted pyridine borane sequencing) with triplicates per sample across laboratories. We observe strand-specific methylation biases across all protocols and libraries. Cross-laboratory reproducibility analyses reveal high quantitative methylation levels agreement (mean Pearson correlation coefficient (PCC) = 0.96) but low detection concordance (mean Jaccard index = 0.36). Using consensus voting, we construct genome-wide quantitative methylation reference datasets serving as ground truth for proficiency testing. Key technical parameters–including mean CpG depth, coverage, and strand consistency–correlate strongly with reference-dependent quality metrics (recall, PCC, and RMSE). Collectively, these resources establish foundational standards for benchmarking emerging epigenomic technologies and analytical pipelines, enabling robust, standardized quality control in research and clinical applications.

Cereblon (CRBN) serves as the substrate recognition subunit of the CRL4-CRBN E3 ubiquitin ligase complex and isthe primary target of immunomodulatory drugs (IMiDs) in cancer therapy. While CRBN mutations occur infrequently in cancer, reduced CRBN expression correlates with poor IMiD response and treatment resistance, suggesting nongeneticregulatory mechanisms may govern CRBN expression. We investigated CRBN expression patterns across multiple cancer types and examined the role of promotermethylation in regulating CRBN expression and its clinical significance. We analyzed CRBN expression patterns using GENT2 database analysis and examined CRBN methylation status indiverse human cancer cell lines using methylation-specific PCR (MSP) and bisulfite genomic sequencing. We analyzedthe clinical significance of CRBN methylation and expression using TCGA and GENT2 databases with Kaplan-Meiersurvival analyses. CRBN expression was significantly downregulated in breast, cervical, head and neck, lung, and skin cancers comparedto normal tissues. We identified a CpG island 1kb upstream of the CRBN transcriptional start site, with cancer celllines exhibiting partial methylation patterns. Bisulfite sequencing confirmed specific CpG methylation sites withinCRBN promoter regions. CRBN promoter hypermethylation significantly correlated with increased mortality risk inbreast, lung, acute myeloid leukemia, and bladder cancers, while low CRBN expression was associated with poorsurvival in breast, lung, brain, and ovarian cancers. Combined low expression and high methylation stronglypredicted poor outcomes in breast and lung cancers. CRBN expression is partially regulated by promoter methylation in specific cancer types, with epigenetic silencingcontributing to treatment resistance and poor prognosis. CRBN methylation and expression serve as important pancancerprognostic biomarkers, highlighting the potential for epigenetic therapies to restore CRBN function andovercome therapeutic resistance.

BackgroundScarce data exist on host gene methylation in oropharyngeal squamous cell carcinomas (OPCs), which are caused by human papillomavirus (HPV) in approximately 40% of cases. We analyzed the methylation status of two host genes involved in cervical carcinogenesis, FAM19A4 and miR124-2, in formalin-fixed paraffin-embedded (FFPE) tissues of primary HPV-driven OPCs (i.e., HPV-positive and p16INK4A-positive), HPV-negative OPCs, head and neck (HN) squamous cell papillomas, and oral rinse-and-gargles (ORGs) from individuals without HN lesions.MethodsA multiplex real-time methylation-specific PCR on bisulfite-converted DNA was employed (PreCursor-M+, Fujirebio). Hypermethylation for each target was expressed as negative/positive based on the ΔΔCt, as well as by the ΔΔCt ratio (2-ΔΔCt).ResultsA total of 70 HPV-driven OPCs (66 HPV16+, 94.3%), 71 HPV-negative OPCs, 12 HN papillomas, and 20 ORGs were evaluated. Six of the 153 FFPE-purified DNA samples (3.9%) yielded invalid results. Hypermethylation for at least one target gene was observed in 56 of 69 valid HPV-driven OPCs (81.2%) and 33 of 67 valid HPV-negative OPCs (49.2%; p < 0.0001). None of the papillomas or ORGs were hypermethylated. HPV-driven OPCs showed a significantly higher methylation level compared with HPV-negative OPCs for both FAM19A4 (median ΔΔCt ratio 11.95 vs. 5.49; p = 0.0001) and miR124-2 (median ΔΔCt ratio 15.65 vs. 8.27; p < 0.0001).ConclusionsOur findings indicate that FAM19A4/miR124-2 hypermethylation occurs exclusively in tumor samples and that methylation levels are significantly higher in HPV-driven OPCs than in HPV-negative OPCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13027-025-00697-5.

SHOX gene haploinsufficiency is associated with Léri-Weill dyschondrosteosis (LWD) or idiopathic short stature (ISS) and could be caused by the structural and point mutations in the coding region and by the deletions in SHOX gene regulatory sequences. The role of the duplications in regulatory sequences is ambivalent. The unanswered question is what the role of changes in SHOX gene methylation is and if they could arise as a result of SHOX area duplication. Material consisted of DNA samples from 20 LWD patients with duplication involving SHOX regulatory elements, 30 patients with LWD/ISS phenotype without any known causal mutation in the SHOX region, and 23 healthy individuals as controls. We investigated the DNA methylation status of two CpG islands in the upstream region of the SHOX using bisulfite sequencing. Our results indicate that both CpG islands in the SHOX area show a lower level of methylation in LWD patients carrying duplications than in healthy individuals, but only one island showed a statistically significant difference. The results of methylation profiling of CpG islands in patients without any known causal mutation indicate that the methylation levels of the majority of patients differed in both CpG islands from the average of the group of healthy individuals by at least ±2 SD. However, the biological effect of these differences will probably be clinically insignificant due to the generally very low level of methylation.

Peritoneal dissemination is a major cause of poor prognosis in gastric cancer (GC). Although conventional peritoneal lavage cytology (CY) is used to detect micrometastatic peritoneal spread, its sensitivity is limited. This study aimed to evaluate the clinical utility of droplet digital methylation-specific PCR (ddMSP) targeting cancer-specific methylation for DNA-based detection of peritoneal dissemination. Peritoneal lavage fluid was prospectively collected from 400 samples in 357 GC patients, including 360 samples from 339 patients with chemotherapy-naïve tumors. DNA was extracted, bisulfite-converted, and analyzed by ddMSP targeting CDO1 and HOPX methylation. Diagnostic performance was assessed by ROC analysis, and associations with clinicopathological features and prognosis were evaluated using logistic and Cox regression models. CDO1 and HOPX methylation levels were significantly elevated in CY1 cases compared with CY0 (p < 0.0001). CDO1 methylation demonstrated excellent diagnostic accuracy for CY1 (AUC: 0.93; sensitivity: 83.9%; specificity: 90.9%), while HOPX methylation showed slightly lower performance (AUC: 0.86). Multivariate analysis revealed that ddMSP CDO1-hi was independently associated with serosal invasion (pT4), and HOPX-hi with both pT4 and nodal metastasis. Furthermore, CDO1-hi status was an independent adverse prognostic factor for peritoneal dissemination-free survival (HR: 2.73, p = 0.018). ddMSP-based detection of CDO1 methylation provides a sensitive and specific method for identifying micrometastatic peritoneal spread in GC. This DNA-based approach may serve as a valuable prognostic biomarker and contribute to improved perioperative management in gastric cancer.

Introduction. Glioblastoma is the most common malignant brain tumor in adults with a poor prognosis. Treatment of patients includes surgical resection, radiation and the alkylating agent temozolomide (TMZ). The therapeutic efficacy of TMZ is due to its ability to damage DNA and induce apoptosis, but it is neutralized by the expression of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT). Methylation of the MGMT gene promoter suppresses the synthesis of the corresponding enzyme and increases the cytotoxic efficiency of TMZ.Aim. To determine the MGMT promoter methylation in glioblastoma patients and to evaluate the prognostic significance of this phenomenon.Materials and methods. Bisulfite-treated DNA samples isolated from formalin-fixed paraffin-embedded tumor tissues obtained from glioblastoma patients were analyzed. MGMT methylation was assessed by qualitative methylation-specific polymerase chain reaction. The prognostic significance of this phenomenon in conjunction with a number of other clinical parameters was assessed by means of univariate and multivariate analysis.Results. MGMT promoter methylation was found to be one of the most significant favorable prognostic factors of glioblastoma: the likelihood of disease reappearance or a fatal outcome at a specific point in time is approximately two-fold lower in such patients than in those with intact MGMT.Conclusion. The methylation-specific polymerase chain reaction, which is routinely used in clinical practice, adequately assesses MGMT methylation status as a prognostic factor, but it does not allow for the evaluation of its predictive potential.

Epigenetic variation contributes to explaining the missing heritability of complex traits. In order to understand the genome-wide methylation variation in spring barley, our objectives were to gain fundamental insight into the barley methylome through whole genome bisulfite sequencing, characterizing methylation variation among 23 parental inbreds of a community resource for genetic mapping of phenotypic traits, and assessing the association of differentially methylated regions (DMRs) with single nucleotide polymorphisms (SNPs) and gene expression variation. Compared to other angiosperms, barley was found to have a highly methylated genome with an average genome wide methylation level of 88.6%, 58.1%, and 1.4% in the CpG, CHG, and CHH sequence context, respectively. We identified just below 500 000 differentially methylated regions (DMRs) among the inbreds. About 64%, 64%, and 83% of the DMRs were not associated with genomic variation in the CpG, CHG, and CHH context, respectively. The methylation level of around 6% of all DMRs was significantly associated with gene expression, where the directionality of the correlation depended on the relative location of the DMR to the respective gene with a recognizable pattern. Notably, this pattern was much more specific and spatially confined than the association of methylation with gene expression across genes in a singular inbred line. We exemplified this association between DNA methylation and gene expression on the known flowering promoting gene VRN-H1 and identified a highly methylated epiallele associated with earlier flowering time. Finally, methylation was shown to improve the prediction abilities of genomic prediction models for a variety of traits over models using solely SNPs and gene expression as predictors. These observations highlight the independence of DNA methylation to sequence variation and their difference in information content. Our discoveries suggest that epigenetic variation provides a layer of information likely not predictable by other means and is therefore a valuable addition to genomic prediction models.

Sexual dimorphism in dogs (Canis lupus familiaris) manifests through pronounced differences in morphology, physiology, and disease susceptibility. Despite early neutering, the persistence of sex-specific differences highlights the need to investigate factors beyond sex hormones that contribute to these characteristics. We collected whole blood tissue from spayed female (n = 4) and castrated male (n = 4) beagles and performed whole genome bisulfite sequencing (WGBS) and RNA seq. To investigate hormone-independent sex dimorphism of DNA methylation in neutered dogs, we investigated differentially methylated genes (DMGs) between sexes and candidate molecular pathways. Furthermore, we analyzed sex-related correlations between gene expression and methylation levels. Sex-related differentially methylated genes, independent of hormone influence, are associated with oncogenic signaling and neuronal pathways. Differences in methylation status between the sexes were significantly associated with alterations in gene expression, indicating that methylation plays a regulatory role in gene transcription. Identification of canine XIST, previously annotated as LOC102156855, suggests a conserved mechanism of X-chromosome inactivation across species and a sex-specific epigenetic imprint on the genome, which is maintained independent of sex hormones. These findings enrich the understanding of sex-specific biology in dogs and highlight the intricate interplay between epigenetic modifications and gene expression in determining sex-specific phenotypes and disease susceptibilities.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19398-5.

Soil salinization is a threat to global agriculture. This study used whole-genome bisulfite sequencing and transcriptomics to explore epigenetic regulation in salt-tolerant Brassica napus (cv. Huayouza 62) under NaCl (salt), Na2CO3 (alkali), and combined salt-alkali stresses. All stresses induced genome-wide DNA hypermethylation (combined > alkali > salt). The Cn subgenome had significant methylation changes (most at CHH, then at CHG), with more differentially methylated regions in transposable elements than protein-coding genes. Hypermethylation, driven by DNA methyltransferases (e.g., DRM2), inhibited growth by repressing photosynthetic genes (RBCS-1A, RBCS-1B). B. napus adapted via targeted demethylation activated stress resistance (ROS scavenging under salt, redox buffering under alkali, and protein homeostasis under combined stress). 5-Azacytidine validation confirmed that methylation balances photosynthesis and stress response, clarifying the epigenetic network and providing strategies for crop stress resistance improvement.

ABSTRACT Background The Igκ locus undergoes multiple molecular processes during B cell development, including V(D)J recombination and epigenetic regulation, which are influenced by cis-regulatory regions within the locus. A novel cis-regulatory region, termed the Dm element, has been identified. It functions in coordination with the 3′Eκ and Ed enhancers and has been implicated in Igκ demethylation and somatic hypermutations (SHM). The Dm element is characterized by a high density of CpG dinucleotides, a hallmark of region subject to DNA methylation. Our previous work demonstrated that RAG2, but not RAG1, contributes to the Igκ locus demethylation. However, whether RAG proteins influence the epigenetic state of the Dm element remains unknown. Methods Here, we investigated the epigenetic state of the Dm element using bisulfite sequencing and chromatin immunoprecipitation (ChIP). Results We found that the Dm element was hypermethylated in pre-B cells but partially demethylated in splenic B cells. Furthermore, it was marked by active histone modifications, including H3K27Ac and H3K9Ac, and was bound by B cell-specific transcription factor Pax5 in pre-B cells. Conclusion Our findings provide evidence that the Dm element undergoes DNA methylation remodeling in mature B cells, potentially contributing to Igκ allelic expression.

BackgroundTo assess the status of NEUROG1 methylation in the advanced adenoma and colorectal cancer.MethodsThe NEUROG1 methylation in tissue and stool samples from patients with colorectal cancer (CRC), advanced adenoma (AA), and non-advanced adenoma (NAA) were evaluated using methylation-specific quantitative polymerase chain reaction (PCR).ResultsIn tissue samples, the NEUROG1 methylation detection rates were 36% for CRC, 24% for NAA, and 88% for AA. In stool samples, the NEUROG1 methylation detection had a sensitivity of 63.46% for CRC with a positive predictive value (PPV) of 85.94%. The overall diagnostic specificity of the test for the NAA and the healthy control was 76.32%, with a negative predictive value (NPV) of 40.28%.ConclusionNEUROG1 methylation detection can potentially be used in the CRC and AA screening.

Environmental changes can induce epigenetic modifications, influencing gene expression, phenotype, and species adaptation. This study investigates how temperature affects genome-wide DNA methylation patterns, particularly in genes crucial for sex development and whether these modifications can be transmitted across generations. Using the European sea bass —a fish model with both genetic and environmental sex determination— we analyzed DNA methylation at single nucleotide resolution using reduced representation bisulfite sequencing in 64 individuals from five families across two generations (F0 and F1). Parental fish (F0) were exposed to either control (16 °C, C) or elevated (21 °C, T) temperatures from 12 to 60 days post-fertilization. Their offspring (F1) were then subjected to four thermal regimes: control (CC), ancestral exposure via sires (TC), developmental exposure in offspring (CT), and dual exposure (TT). We determined the length of differentially methylated regions (DMRs) using a conservative, reproducible, and species-specific method adapted from plant epigenetics. To disentangle ancestral and developmental temperature effects, DMRs were classified according to their association with F0, F1, or F0 x F1 interaction effects. This allowed us to quantify the relative contribution of each treatment, separately for testes and ovaries in the F1 generation. While the proportion of additive DMRs showing cumulative temperature effects (e.g., 2.1% in testes, 1.4% in ovaries) was relatively rare, a substantial proportion of DMRs (37% in testes, 31.1% in ovaries), exhibited opposing methylation changes with F0 and F1 treatments, indicative of compensatory epigenetic interactions. These interactions were also reflected at the phenotypic level: TT individuals showed body weights comparable to CC, and the sex ratio in TT approached statistical significance when compared to CC (P = 0.051), suggesting a link between epigenetic regulation and phenotypic plasticity under elevated temperatures. Finally, we also investigated the inheritance of epimarks from sires to offspring. While most epimarks remained stable across generations, ~ 5% of all DMRs were both temperature-induced and inherited, offering direct evidence for environmentally responsive multigenerational epigenetic inheritance. This study demonstrates the role of temperature in shaping the epigenome and highlights the potential of epigenetic plasticity and inheritance in species adaptation and conservation amid global warming.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-025-00630-5.

Adipose inflammation plays a key role in obesity-induced metabolic abnormalities. Epigenetic regulation, including DNA methylation, is a molecular link between environmental factors and complex diseases. Here we found that high-fat diet (HFD) feeding induced a dynamic change of DNA methylome in mouse white adipose tissue (WAT) analyzed by reduced representative bisulfite sequencing. Interestingly, DNA methylation at the promoter of estrogen receptor α (Esr1) was significantly increased by HFD, concomitant with a downregulation of Esr1 expression. HFD feeding in mice increased the expression of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and binding of DNMT1 and DNMT3a to Esr1 promoter in WAT. Mice with adipocyte-specific Dnmt1 deficiency displayed increased Esr1 expression, decreased adipose inflammation, and improved insulin sensitivity upon HFD challenge; mice with adipocyte-specific Dnmt3a deficiency showed a mild metabolic phenotype. Using a modified CRISPR/RNA-guided system to specifically target DNA methylation at the Esr1 promoter in WAT, we found that reducing DNA methylation at Esr1 promoter increased Esr1 expression, decreased adipose inflammation, and improved insulin sensitivity in HFD-challenged mice. Our study demonstrated that DNA methylation at Esr1 promoter played an important role in regulating adipose inflammation, which may contribute to obesity-induced insulin resistance.

Aberrant DNA methylation and copy number alterations (CNAs) drive Barrett's esophagus (BE) progression to esophageal adenocarcinoma (EAC); however, their combined utility for early detection is unclear. We aimed to identify and validate methylated DNA markers (MDMs) and CNAs to distinguish EAC/high-grade dysplasia (HGD) from non-dysplastic Barrett's esophagus (NDBE). In this multi-phase, multi-center study, we discovered and validated MDMs and quantified CNAs utilizing whole-genome methylation sequencing of esophageal brushings. DNA biomarkers identified from discovery were further validated in independent patients with paired esophageal brushing and swallowed capsule sponge samples. MDMs were filtered against a reduced representation bisulfite sequencing data set obtained from independent tissue samples to advance only concordant candidates. CNA burden was quantified using ichorCNA-derived aneuploidy scores (AS). Two hundred MDMs discovered in HGD (N=18) and EAC (N=18) versus NDBE brushing samples (N=18) were tested in independent samples (N=146). A 52-MDM panel achieved a cross-validated area under the receiver operating characteristic curve (AUC) 0.88 (95% CI: 0.82-0.95); the addition of AS improved discrimination of HGD/EAC from NDBE to 0.91 (95% CI: 0.86-0.97) AUC. At 80% specificity, the combined model detected 93% of EAC and 88% of HGD cases. In paired capsule sponge samples, a 58-MDM panel achieved a cross-validated AUC of 0.77 (95% CI: 0.66-0.88); a combined 58-MDM and AS model achieved AUC 0.80 (95% CI: 0.7-0.9). MDMs and AS discerned HGD/EAC from normal esophagus (NE)/NDBE in endoscopic brushing and capsule sponge samples. This approach may improve BE surveillance.

DNA methylation, a key epigenetic modification, influences gene expression, represses transposable elements, and mediates plant responses to environmental stress by adding methyl groups to cytosine residues (5mC) in CG, CHG, and CHH contexts. There are several methods for locus-specific DNA methylation analysis, each with its own advantages and limitations, including bisulfite sequencing PCR (BSP), methylation-specific PCR (MSP), bisulfite pyrosequencing, epityper, methylation-sensitive restriction enzyme (MSRE)-based approaches such as MSRE-Southern blot and MSRE-PCR, methylation-dependent restriction enzyme methods like McrBC, and Affinity Enrichment-based techniques. Here, we present a detailed protocol for locus-specific DNA methylation analysis, highlighting sodium bisulfite sequencing as the gold standard due to its single-base resolution, simplicity, and high accuracy. This protocol utilizes the EZ DNA Methylation-Gold™ Kit for bisulfite conversion, which selectively deaminates unmethylated cytosines to uracil while preserving methylated cytosines. The procedure involves treatment with the CT conversion reagent, followed by thermal cycling and purification using Zymo-Spin™ IC columns. To ensure specific and unbiased amplification of bisulfite-converted DNA, primers are carefully designed using tools such as MethPrimer and BiSearch. Following bisulfite conversion and PCR amplification of the target region, the amplified products are cloned into a binary vector and subjected to Sanger sequencing, enabling single-base resolution analysis of DNA methylation patterns. This approach allows for precise mapping of DNA methylation at promoters, enhancers, and transposable elements, providing valuable insights into gene regulation and phenotypic variation. Locus-specific DNA methylation analysis in plants has wide-ranging applications, including the study of epigenetic responses to stress, transgenerational inheritance, and crop improvement.

A subset of cytosine bases in plant nuclear genomes is methylated at the fifth position, forming 5-methylcytosine (5mC). DNA methylation is a conserved epigenetic mark important for genome stability and gene transcription. The genome methylation status is altered under conditions of stress, affecting gene expression patterns as a resistance response. Furthermore, changes in genome methylation status can be transmitted to germline cells in many plant species by overcoming the restraints on meiosis, resulting in the inheritance of acquired resistance traits in the next generation. Arabidopsis (Arabidopsis thaliana) is an attractive research model plant for studies of transgenerational inheritance due to its short life cycle (8-10weeks). Isogenic lines of plants should be used throughout a transgenerational inheritance study to avoid genetic differences between individuals in the descendant generations. In addition, plants should be germinated and grown under optimal conditions to minimize environmental factors that may cause DNA methylome variations between the generations. 5mC in genomic DNA can be experimentally distinguished from unmethylated cytosine by treatment with sodium bisulfite, which converts unmethylated cytosine, but not 5mC, to uracil. Genomic regions with differential methylation between generations can be identified via whole-genome bisulfite sequencing and alignment of sequences between generations. Apart from whole-genome sequencing, particular chromatin regions of interest can be amplified by methylation-specific polymerase chain reaction (PCR). In this chapter, experimental steps for studying transgenerational inheritance of gene methylation are described using Arabidopsis as a model plant species.

Tumor heterogeneity has been recognized as a critical characteristic of Ovarian Cancer (OC), significantly influencing chemotherapeutic response and overall patient survival. The VEGF/VEGFR2 signaling pathway genes serve as risk factor for metastasis and are associated with poor prognosis in many cancers including OC. DNA methylation of VEGF-A, VEGF-C and VEGFR2 genes contributing to their genetic changes based on histological sub-types has not been thoroughly explored in OC. Thus, the study aimed to determine the DNA methylation status of these genes in relation to their expression in tumor tissues and associated peritoneal fluid samples to evaluate usefulness of methylation as a prognostic indicator in ovarian carcinoma for future targeted therapies. Bisulfite-modified DNA methylation was assessed via Methylation-Specific Polymerase chain reaction (MSP) and mRNA expression was examined via quantitative reverse transcription PCR (RT-PCR), respectively. Gene expression and promoter methylation were examined in relation with clinical parameters in 100 OC samples. The methylation frequency of VEGF-A, VEGF-C and VEGFR2 promoters was higher in tumor tissues and matched Peritoneal Fluid (PF) compared to normal samples, while VEGF-A, VEGF-C and VEGFR2 mRNA expression levels were significantly elevated. Promoter methylation levels for VEGF-A (82%), VEGF-C (76%) and VEGFR2 (82%) showed significantly higher levels compared to benign cystadenomas, whereas amounts of VEGF-A and VEGFR2 were significantly higher than benign samples (p = 0.0003 and 0.001). Expression and promoter methylation for VEGF-A (p = 0.0004, 0.0001) and VEGFR2 (p = 0.005, 0.001) significantly correlated with differences in histological sub-types in OC samples. No significant correlation was observed between promoter methylation and expression levels for VEGF-A, VEGF-C and VEGFR2 genes. Kaplan-Meier survival analysis predicts poor prognosis for concurrent high expression and methylation of VEGF-A and VEGFR2 significantly correlated with advanced stage (log-rank p = 0.03, 0.01) and tumor histology (log rank p = 0.02, 0.01 respectively) in OC patients. Promoter methylation status of the VEGF-A and VEGFR2 genes could serve as valuable prognostic indicator in predicting poor prognosis in OC patients based on tumor histological sub-types.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-04483-6.

Oral squamous cell carcinoma (OSCC) is a major health burden in most parts of the world, and pathogenesis of the disease is strongly associated with a complex combination between genetic mutations and epigenetic alterations. The current study will explore the DNA methylation phenomenon and how it affects the silencing of tumour suppressor genes and activate oncogenes in OSCC in a bid to explain the driving molecular process behind tumour development. We evaluated 50 samples of the OSCC tissues with 50 of the adjacent normal tissues, evaluated DNA methylation patterns with methylation-specific PCR and quantitative reverse-transcriptase PCR measured the expression of the DNA repair genes like BRCA1 and MLH1, among others. This finding showed different levels of DNA methylation of cancerous and normal tissues with hypermethylation causing the inactivation of important tumor suppressor genes and hypomethylation causing the activation of oncogenes. In addition, the downregulation of DNA repair genes was noted to be highly significant in OSCC samples indicating that genomic-instability may be related to epigenetic changes. These results demonstrate that aberrant DNA methylation is central to OSCC growth and progression, thus helping in the future use of methylation patterns that can be used as early detection, diagnosis, and prognostic biomarkers. Our findings support the idea that genetic, along with epigenetic, profiling is an issue of key importance toward comprehending OSCC biology and personalized therapeutic interventions. It is recommended that further confirmation be carried out to verify the clinical significance of such epigenetic markers in bigger cohorts, as well as testing such markers in guided therapies, which might eventually lead to better patient outcomes in the management of oral cancer.

DNA methylation is a major epigenetic mark on chromatin. Tissue-specific DNA methylation in the regulatory regions of key genes modulates their tissue-specific expression. To study DNA methylation levels at specific sequence contexts across tissues or during various environmental conditions, whole-genome DNA methylation analysis is widely used. However, whole-genome methylation analysis is an expensive, time-consuming technique and requires specific skills. To understand the role of DNA methylation in a small number of candidate loci, targeted bisulfite (BS) PCR-sequencing analysis is an attractive option. Here, we describe a detailed method to understand DNA methylation by targeted-BS PCR analysis using rice endosperm tissues. This method can be readily extended to multiple monocots, across various tissues and various stress conditions.