Hypermethylation Of Region Research Articles

Epigenetics and cancer: the role of DNA methylation

Alterations in DNA methylation profiles are typically found in cancer cells, combining genome-wide hypomethylation with hypermethylation of specific regions, such as CpG islands, which are normally unmethylated. Driving effects in cancer development have been associated with alteration of DNA methylation in certain regions, inducing, for example, the repression of tumor suppressor genes or the activation of oncogenes and retrotransposons. These alterations represent prime candidates for the development of specific markers for the detection, diagnosis and prognosis of cancer. In particular, these markers, distributed along the genome, provide a wealth of information that offers potential for innovation in the field of liquid biopsy, in particular thanks to the emergence of artificial intelligence for diagnostic purposes. This could overcome the limitations related to sensitivities and specificities, which remain too low for the most difficult applications in oncology: the detection of cancers at an early stage, the monitoring of residual disease and the analysis of brain tumors. In addition, targeting the enzymatic processes that control the epigenome offers new therapeutic strategies that could reverse the regulatory anomalies of these altered epigenomes.

Key Epigenetic Players in Etiology and Novel Combinatorial Therapies for Treatment of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of death in which the molecular tumorigenesis and cellular heterogeneity are poorly understood. The genetic principle that specific driver mutations in oncogenes, DNA repair genes, and tumor-suppressor genes can independently drive cancer development has been widely explored. Additionally, a repertory of harmful epigenetic modifications in DNA and chromatin—impacting the expression of genes involved in cellular proliferation, differentiation, genome stability, cell-cycle control, and DNA repair—are now acknowledged across various biological contexts that contribute to cancer etiology. Notably, the dynamic hypermethylation and hypomethylation in enhancer and promoter regions that promote activation or silencing of the master regulatory genes of the epigenetic programs is often altered in tumor cells due to mutation. Genome instability is one of the cancer hallmarks that contribute to transdifferentiation and intratumoral heterogeneity. Thus, it is broadly accepted that tumor tissue is dominated by genetically and epigenetically distinct sub-clones which display a set of genetic and epigenetic mutations. Here we summarize some functions of key genetic and epigenetic players and biochemical pathways leading to liver cell transformation. We discuss the role of the potential epigenetic marks in target genes thought to be involved in sequential events following liver lipid metabolism dysregulation, inflammation, fibrosis, cirrhosis, and finally hepatocellular carcinoma. We also briefly describe new findings showing how epigenetic drugs together with chemotherapy and immunotherapy can improve overall responses in patients with hepatic tumors.

Distinct epigenetic and transcriptional profiles of Epstein-Barr virus (EBV) positive and negative primary CNS lymphomas.

Epstein-Barr virus (EBV)+ and EBV- primary CNS lymphomas (PCNSL) carry distinct mutational landscapes, but their transcriptional and epigenetic profiles have not been integrated and compared. This precludes further insights into pathobiology and molecular differences, relevant for classification and targeted therapy. 23 EBV- and 15 EBV+ PCNSL, histologically classified as diffuse large B-cell lymphomas, were subjected to RNA-Sequencing and EPIC methylation arrays. Unsupervised clustering analyses were performed. Differentially expressed and differentially methylated genes were identified and integrated. Two distinct transcriptional clusters were found, which separated EBV-and EBV+PCNSL (p < 0.0001). The EBV+ transcriptional signature contained genes (GPR15, FCER2/CD23, SLAMF1/CD150) closely regulated by EBV oncogenes in B-cells. Pathway enrichment analysis uncovered enhanced B-cell receptor (BCR) and WNT/beta-catenin signaling in EBV-lymphomas, whereas Interleukin-10, NOTCH, and viral life cycle pathways were upregulated in EBV+PCNSL. Correspondingly, BCR-associated SYK kinase activity was enriched in EBV-tumors while JAK2 was overrepresented in EBV+PCNSL. Epigenetic profiling revealed reduced global promoter methylation in EBV+PCNSL. Two methylation clusters were recognized, which separated EBV-and EBV+PCNSL (p < 0.0001). Epigenetic profiles were distinct from 2,788 other brain tumor and non-malignant reference samples. Promoter region hypermethylation of CD79B, a BCR subunit critical for sustained proliferation in EBV-disease, highly correlated (R = -0.7) with its transcriptional downregulation in EBV+PCNSL. EBV+ and EBV- PCNSL harbor distinct transcriptional and epigenetic profiles, corroborating them as distinctive biological subtypes. Uncovered differences provide novel insights into their pathobiology, may guide molecular diagnostics and targeted therapies.

Transcriptomic era of cancers in females: new epigenetic perspectives and therapeutic prospects.

In the era of transcriptomics, the role of epigenetics in the study of cancers in females has gained increasing recognition. This article explores the impact of epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA, on cancers in females, including breast, cervical, and ovarian cancers (1). Our findings suggest that these epigenetic markers not only influence tumor onset, progression, and metastasis but also present novel targets for therapeutic intervention. Detailed analyses of DNA methylation patterns have revealed aberrant events in cancer cells, particularly promoter region hypermethylation, which may lead to silencing of tumor suppressor genes. Furthermore, we examined the complex roles of histone modifications and long non-coding RNAs in regulating the expression of cancer-related genes, thereby providing a scientific basis for developing targeted epigenetic therapies. Our research emphasizes the importance of understanding the functions and mechanisms of epigenetics in cancers in females to develop effective treatment strategies. Future therapeutic approaches may include drugs targeting specific epigenetic markers, which could not only improve therapeutic outcomes but also enhance patient survival and quality of life. Through these efforts, we aim to offer new perspectives and hope for the prevention, diagnosis, and treatment of cancers in females.

Induction of FAM46C expression mediated by DNMT3A downregulation is involved in early-onset preeclampsia through gene body methylation

BackgroundAberrant methylation of genomic DNA has been found in preeclamptic placentas, which is characterized by elevated DNA methylation and hypermethylation of gene body regions, but the underlying mechanism is not yet fully understood. MethodsGlobal DNA methylation was assessed through ELISA and HPLC. The methylation sites were detected using the Illumina Human Methylation 450 K Microarray. The methylation level of FAM46C promoter and gene body was detected through the bisulfite sequencing. RNA-seq was utilized to investigate the mechanism by which DNMT3A and FAM46C mediate the migration and invasion of trophoblast cells. ResultsWe discovered that DNMT3A knockdown led to elevated levels of gene body methylation and FAM46C transcription. FAM46C downregulation completely rescued the suppressive effects caused by DNMT3A knockdown on the migration and invasion of trophoblast cells. Mechanistically, DNMT3A reduction led to an increase in the enrichment of DNMT3B and DNMT1 in the gene body region of FAM46C. The results of transcriptome sequencing showed that DNMT3A and FAM46C regulate the adhesion of trophoblast cells. Elevated expression of FAM46C and increased methylation levels within its gene body region were observed in extravillous trophoblast cells of early-onset preeclamptic placentas. ConclusionsDNMT3A-mediated aberrant FAM46C gene body methylation is relevant to the development of early-onset preeclampsia.

DNA methylation drives hematopoietic stem cell aging phenotypes after proliferative stress.

Aging of hematopoietic stem cells (HSCs) is implicated in various aging phenotypes, including immune dysfunction, anemia, and malignancies. The role of HSC proliferation in driving these aging phenotypes, particularly under stress conditions, remains unclear. Therefore, we induced forced replications of HSCs in vivo by a cyclical treatment with low-dose fluorouracil (5FU) and examined the impact on HSC aging. Our findings show that proliferative stress induces several aging phenotypes, including altered leukocyte counts, decreased lymphoid progenitors, accumulation of HSCs with high expression of Slamf1, and reduced reconstitution potential, without affecting stem cell self-renewal capacity. The divisional history of HSCs was imprinted in the DNA methylome, consistent with functional decline. Specifically, DNA methylation changes included global hypermethylation in non-coding regions and similar frequencies of hypo- and hyper-methylation at promoter regions, particularly affecting genes targeted by the PRC2 complex. Importantly, initial forced replication promoted DNA damage repair accumulated with age, but continuous proliferative stress led to the accumulation of double-strand breaks, independent of functional decline. Overall, our results suggest that HSC proliferation can drive some aging phenotypes primarily through epigenetic mechanisms, including DNA methylation changes.

Comprehensive in silico CpG methylation analysis in hepatocellular carcinoma identifies tissue- and tumor-type specific marks disconnected from gene expression

DNA methylation is crucial for chromatin structure, transcription regulation and genome stability, defining cellular identity. Aberrant hypermethylation of CpG-rich regions is common in cancer, influencing gene expression. However, the specific contributions of individual epigenetic modifications to tumorigenesis remain under investigation. In hepatocellular carcinoma (HCC), DNA methylation alterations are documented as in other tumor types. We aimed to identify hypermethylated CpGs in HCC, assess their specificity across other tumor types, and investigate their impact on gene expression. To this end, public methylomes from HCC, other liver diseases, and 27 tumor types as well as expression data from TCGA-LIHC and GTEx were analyzed. This study identified 39 CpG sites that were hypermethylated in HCC compared to control liver tissue, and were located within promoter, gene bodies, and intergenic CpG islands. Notably, these CpGs were predominantly unmethylated in healthy liver tissue and other normal tissues. Comparative analysis with 27 other tumors revealed both common and HCC-specific hypermethylated CpGs. Interestingly, the HCC-hypermethylated genes showed minimal expression in the different healthy tissues, with marginal changes in the level of expression in the corresponding tumors. These findings confirm previous evidence on the limited influence of DNA hypermethylation on gene expression regulation in cancer. It also highlights the existence of mechanisms that allow the selection of tissue-specific methylation marks in normally unexpressed genes during carcinogenesis. Overall, our study contributes to demonstrate the complexity of cancer epigenetics, emphasizing the need of better understanding the interplay between DNA methylation, gene expression dynamics, and tumorigenesis.Graphical

Epigenetics of Dietary Phytochemicals in Cancer Prevention: Fact or Fiction.

Cancer development takes 10 to 50 years, and epigenetics plays an important role. Recent evidence suggests that ~80% of human cancers are linked to environmental factors impinging upon genetics/epigenetics. Because advanced metastasized cancers are resistant to radiation/chemotherapeutic drugs, cancer prevention by relatively nontoxic "epigenetic modifiers" will be logical. Many dietary phytochemicals possess powerful antioxidant and anti-inflammatory properties that are hallmarks of cancer prevention. Dietary phytochemicals can regulate gene expression of the cellular genome via epigenetic mechanisms. In this review, we will summarize preclinical studies that demonstrate epigenetic mechanisms of dietary phytochemicals in skin, colorectal, and prostate cancer prevention. Key examples of the importance of epigenetic regulation in carcinogenesis include hypermethylation of the NRF2 promoter region in cancer cells, resulting in inhibition of NRF2-ARE signaling. Many dietary phytochemicals demethylate NRF2 promoter region and restore NRF2 signaling. Phytochemicals can also inhibit inflammatory responses via hypermethylation of inflammation-relevant genes to block gene expression. Altogether, dietary phytochemicals are excellent candidates for cancer prevention due to their low toxicity, potent antioxidant and anti-inflammatory properties, and powerful epigenetic effects in reversing procarcinogenic events.

Inflammatory breast cancer microenvironment repertoire based on DNA methylation data deconvolution reveals actionable targets to enhance the treatment efficacy

BackgroundAlthough the clinical signs of inflammatory breast cancer (IBC) resemble acute inflammation, the role played by infiltrating immune and stromal cells in this aggressive disease is uncharted. The tumor microenvironment (TME) presents molecular alterations, such as epimutations, prior to morphological abnormalities. These changes affect the distribution and the intricate communication between the TME components related to cancer prognosis and therapy response. Herein, we explored the global DNA methylation profile of IBC and surrounding tissues to estimate the microenvironment cellular composition and identify epigenetically dysregulated markers.MethodsWe used the HiTIMED algorithm to deconvolve the bulk DNA methylation data of 24 IBC and six surrounding non-tumoral tissues (SNT) (GSE238092) and determine their cellular composition. The prognostic relevance of cell types infiltrating IBC and their relationship with clinicopathological variables were investigated. CD34 (endothelial cell marker) and CD68 (macrophage marker) immunofluorescence staining was evaluated in an independent set of 17 IBC and 16 non-IBC samples.ResultsWe found lower infiltration of endothelial, stromal, memory B, dendritic, and natural killer cells in IBC than in SNT samples. Higher endothelial cell (EC) and stromal cell content were related to better overall survival. EC proportions positively correlated with memory B and memory CD8+ T infiltration in IBC. Immune and EC markers exhibited distinct DNA methylation profiles between IBC and SNT samples, revealing hypermethylated regions mapped to six genes (CD40, CD34, EMCN, HLA-G, PDPN, and TEK). We identified significantly higher CD34 and CD68 protein expression in IBC compared to non-IBC.ConclusionsOur findings underscored cell subsets that distinguished patients with better survival and dysregulated markers potentially actionable through combinations of immunotherapy and epigenetic drugs.

DNA Methylation Profiling in Genetically Selected Clarias magur (Hamilton, 1822) Provides Insights into the Epigenetic Regulation of Growth and Development.

DNA methylation is an epigenetic alteration that impacts gene expression without changing the DNA sequence affecting an organism's phenotype. This study utilized a reduced representation bisulfite sequencing (RRBS) approach to investigate the patterns of DNA methylation in genetically selected Clarias magur stocks. RRBS generated 249.22 million reads, with an average of 490,120 methylation sites detected in various parts of genes, including exons, introns, and intergenic regions. A total of 896 differentially methylated regions (DMRs) were identified; 356 and 540 were detected as hyper-methylated and hypo-methylated regions, respectively. The DMRs and their association with overlapping genes were explored using whole genome data of magur, which revealed 205 genes in exonic, 210 in intronic, and 480 in intergenic regions. The analysis identified the maximum number of genes enriched in biological processes such as RNA biosynthetic process, response to growth factors, nervous system development, neurogenesis, and anatomical structure morphogenesis. Differentially methylated genes (DMGs) such as myrip, mylk3, mafb, egr3, ndnf, meis2a, foxn3, bmp1a, plxna3, fgf6, sipa1l1, mcu, cnot8, trim55b, and myof were associated with growth and development. The selected DMGs were analyzed using real-time PCR, which showed altered mRNA expression levels. This work offers insights into the epigenetic mechanisms governing growth performance regulation in magur stocks. This work provides a valuable resource of epigenetic data that could be integrated into breeding programs to select high-performing individuals.

RNA sequencing analysis of sexual dimorphism in Japanese quail.

Japanese quail are of significant economic value, providing protein nutrition to humans through their reproductive activity; however, sexual dimorphism in this species remains relatively unexplored compared with other model species. A total of 114 RNA sequencing datasets (18 and 96 samples for quail and chicken, respectively) were collected from existing studies to gain a comprehensive understanding of sexual dimorphism in quail. Cross-species integrated analyses were performed with transcriptome data from evolutionarily close chickens to identify sex-biased genes in the embryonic, adult brain, and gonadal tissues. Our findings indicate that the expression patterns of genes involved in sex-determination mechanisms during embryonic development, as well as those of most sex-biased genes in the adult brain and gonads, are identical between quails and chickens. Similar to most birds with a ZW sex determination system, quails lacked global dosage compensation for the Z chromosome, resulting in directional outcomes that supported the hypothesis that sex is determined by the individual dosage of Z-chromosomal genes, including long non-coding RNAs located in the male hypermethylated region. Furthermore, genes, such as WNT4 and VIP, reversed their sex-biased patterns at different points in embryonic development and/or in different adult tissues, suggesting a potential hurdle in breeding and transgenic experiments involving avian sex-related traits. The findings of this study are expected to enhance our understanding of sexual dimorphism in birds and subsequently facilitate insights into the field of breeding and transgenesis of sex-related traits that economically benefit humans.

Quantification method of ctDNA using cell-free DNA methylation profile for noninvasive screening and monitoring of colon cancer

BackgroundColon cancer ranks as the second most lethal form of cancer globally. In recent years, there has been active investigation into using the methylation profile of circulating tumor DNA (ctDNA), derived from blood, as a promising indicator for diagnosing and monitoring colon cancer.ResultsWe propose a liquid biopsy-based epigenetic method developed by utilizing 49 patients and 260 healthy controls methylation profile data to screen and monitor colon cancer. Our method initially identified 901 colon cancer-specific hypermethylated (CaSH) regions in the tissues of the 49 cancer patients. We then used these CaSH regions to accurately quantify the amount of circulating tumor DNA (ctDNA) in the blood samples of these same patients, utilizing cell-free DNA methylation profiles. Notably, the methylation profiles of ctDNA in the blood exhibited high sensitivity (82%) and specificity (93%) in distinguishing patients with colon cancer from the control group, with an area under the curve of 0.903. Furthermore, we confirm that our method for ctDNA quantification is effective for monitoring cancer patients and can serve as a valuable tool for postoperative prognosis.ConclusionsThis study demonstrated a successful application of the quantification of ctDNA among cfDNA using the original cancer tissue-derived CaSH region for screening and monitoring colon cancer.

Differential expression of long non-coding RNAs in colon cancer: Insights from transcriptomic analysis

BackgroundColon Cancer (CC) incidence has sharply grown in recent years. Long non-coding RNAs (lncRNA) are produced by a group of non-protein-coding genes, and have important functions in controlling gene expression and impacting the biological features of various malignancies including CC. MethodsOur research focused on examining the function of lncRNAs in the development of colon cancer. To this end, we selected and analyzed a dataset (GSE104836) from the GEO database, which contained information about the expression of mRNAs and lncRNAs in both colon cancer tissues and normal adjacent paired tumor tissues. The DESeq2 R package in Bioconductor was used to identify differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) that showed differences in expression levels. Next, by literature review of previous studies, we chose two lncRNAs (FENDRR and LINC00092) for additional studies. To validate our findings, a series of tests were performed on a total of 31 tumor tissues and normal paired adjacent tumor tissues. The lncRNA expression levels were assessed in tumor tissues as well as in surrounding normal tumor tissues. ResultsThe data confirmed that just two particular lncRNAs, FENDRR and LINC00092, had considerably decreased expression levels throughout all stages of cancer. In addition, the survival assay was conducted using the GEPIA2 software, revealing that a reduced expression of FENDRR is correlated with a reduced overall survival. Furthermore, our investigation using receiver operating characteristic (ROC) methodology revealed that these two lncRNAs had significant discriminatory ability between colon cancer and normal tissues. To determine the cause of the decrease in the activity of these two long non-coding RNAs (lncRNAs), we used methylation-specific PCR (MSP) to examine the methylation pattern of their promoter regions. Our investigation revealed hypermethylation in the promoter regions of FENDRR and LINC00092 within tumor tissues compared to normal adjacent tumor tissues. ConclusionTaken together, our findings revealed the lncRNAs signatures as potential therapeutic targets and molecular diagnostic biomarkers in colon cancer. Furthermore, the evidence provided substantiates the important role of promoter methylation in regulating the expression levels for both of these lncRNAs.

Combining non-invasive liquid biopsy and a methylation analysis to assess surgical risk for early esophageal cancer.

While the widespread use of endoscopic submucosal dissection (ESD) has significantly reduced the incidence of early esophageal cancer (ESCA), the limited ability of ESD to strip deep infiltrating esophageal lesions results in a considerable risk of intraoperative perforation. Circulating-free DNA (cfDNA) is widely used in modern tumor screening due to its non-invasive detection capabilities. A methylation analysis offers vital insights into the condition and advancement of malignancies due to its unique positioning, such as a marker of cancer. This study investigated the potential of combining a non-invasive liquid biopsy technique, along with a methylation analysis, to assess the surgical perforation risk of ESCA patients. In this study, we conducted an analysis of gene expression differences between stage I esophageal squamous carcinoma samples and healthy tissue samples using data from The Cancer Genome Atlas (TCGA) database. We also identified the genes associated with progression-free survival (PFS) in esophageal squamous carcinoma. Integrating the framework of the methylation analysis, we explored the methylated sites of these distinct genes. To refine this process, we used the Shiny Methylation Analysis Resource Tool (SMART) to conduct a comprehensive analysis of these sites. We then confirmed the stability of the methylation sites in different lesion conditions using methylation-specific quantitative polymerase chain reaction (MS-qPCR) with paraffin tissue samples collected after ESD. We analyzed RNA-sequencing data from 42 early stage ESCA patients and 17 controls, identifying 1,263 up-regulated and 460 down-regulated genes. Functional analyses revealed involvement in key pathways such as cell cycle regulation and immune responses. Furthermore, we identified 38 differentially expressed genes associated with PFS. Using SMART analysis, we found 217 hyper-methylated regions in 38 genes, suggesting potential early markers for ESCA. Validation experiments confirmed the reliability of 29 hyper-methylated regions in FFPE tissue samples and 6 regions in cfDNA. A LunaCAM model showed high accuracy [area under the curve (AUC) =0.89] in discriminating early ESCA. Integrated assessment of six highly methylated regions significantly improved predictive performance, with 90.56% sensitivity, highlighting the importance of combinatorial biomarker evaluation for early cancer detection. This study established a novel approach that integrates non-invasive testing with a methylation analysis to assess the surgical risk of early ESCA patients. The significance of changes in methylation sites in relation to lesion status should not be underestimated, as they have the potential to offer vital insights for proactive risk assessments before surgery.

Genetic-epigenetic aspects of infertility in women with long-COVID-19

The aim of the study is to determine the genetic and epigenetic features of infertile patients with long-term COVID-19 in order to clarify the risk factors for the failure of assisted reproductive technologies (ART) use. Materials and methods. The MTHFR (C677T, A1298С), RFC-1 (G80A) and BHMT (G742A) gene polymorphisms and the methylation of ESR1 gene was performed. 40 patients (main group) with infertility due to long-term COVID-19 were examined: the subgroup 1 - 25 women in whom the use of ART was ineffective, the subgroup 2 - 15 patients with effective use of ART. The control was taken from literary sources: for RFC (G80A), MTHFR (C677T) and MTHFR (A1298C) polymorphisms - 35 Ukrainian women without infertility and reproductive losses; for BHMT (G742A) - 60 people of the Ukrainian population. The methods of variational statistics were used, in particular the Fisher test with a significance level of p&lt;0.05. Results. Patients of the main group have a higher frequency of the homozygous genotype of the MTHFR gene polymorphism (C677T) on the mutant T allele (20.0% vs. 3.2%; p&lt;0.05). When ART is unsuccessful, the TT genotype is 4 times greater (28.0% vs. 6.7%; p&lt;0.05). A study by genotypes of MTHFR polymorphism (A1298C) did not reveal a significant difference. Patients of the main group `have a higher frequency of the mutant allele A of the RFC gene (G80A) (80.0% vs. 51.4%; p&lt;0.05). No significant difference was found depending on the success rate of ART. The frequency of BHMT gene polymorphism (G742A) in the main group did not differ from that in the Ukrainian population, however, in the case of unsuccessful ART, it was observed less often and only in the heterozygous variant (40.0% vs. 66.6%; p&lt;0.05). The analysis of pairwise intergenic interaction revealed the highest frequency of the combination of AAGA for the MTHFR (A1298C)+BHMT (G742A) pair - 35.0% and GAAA for the RFC (G80A)+MTHFR (A1298C) pair - 30.0% in the main group. Hypermethylation of the promoter region of the ESR1 gene is observed in 20 (50.0%) patients of the main group: in 17 (68.0%) women of the subgroup 1 versus 3 (20.0%) women of the subgroup 2 (p&lt;0.05). Conclusions. The genetic and epigenetic conditioning of the success of ART programs in infertility associated with long-term COVID-19 has been revealed, which opens up new diagnostic and therapeutic opportunities for identifying factors predisposing to unsuccessful ART treatment and increasing the effectiveness of such treatment by correcting disorders of folic acid metabolism, hyperhomocysteinemia and the estrogens receptor apparatus. The research was carried out in accordance with the principles of the Declaration of Helsinki. The research protocol was approved by the Local Ethics Committee of the institution mentioned in the work. Informed consent of the women was obtained for the research. The authors declare no conflict of interest.

Non-invasive diagnosis of nature of pulmonary nodules using the whole genome methylation sequencing of circulating cell-free DNA.

e20038 Background: The frequent false-positive results of lung cancer screening of low-dose computed tomography (LDCT) remains a challenge. In this study, we aimed to establish a novel panel of cancer-specific methylated regions detected from circulating cell-free DNA (cfDNA) for differentiating malignant pulmonary nodules (PNs) from benign lesions in high-risk patients. Methods: We performed DNA methylation profiling by high throughput DNA bisulfite sequencing in tissue samples (nodule size &lt; 3 cm in diameter) to learn methylation patterns that differentiate cancerous tumors from benign lesions. Then we filtered out differential methylation patterns in circulating cfDNA that are consistent with tissue expression patterns and built an assay for plasma sample classification. Area under the curve was computed for each receiving operation curve, and 95% confidence intervals were also estimated by bootstrapping with 1,000 iterations. Results: We first performed the whole genome methylation sequencing of 100 tissue samples from PNs to learn cancer-specific methylation patterns, and obtained 2,406 differential hypermethylated regions and 16,028 differential hypomethylated regions. These tissue-derived DNA methylation markers were further filtered using a training set of 42 plasma samples and 11 markers were ultimately identified to build a diagnostic prediction model. This methylation panel provided promising results in an independent validation set of additional 17 plasma samples, with a sensitivity of 66.67% (95% CI: 0.25–1.00) and a specificity of 79.17% for differentiating patients with malignant tumor (N = 12) from patients with benign lesions (n = 5). A further validation study of this methylation panel in the cancer genome atlas data set demonstrated significant discriminatory performance in distinguishing patients with lung cancer from subjects without malignancy (area under the curve = 0.88). Conclusions: Our model based on the measurement of circulating cfDNA methylation provide a minimally invasive procedure for differentiation of early-stage lung cancer and nonmalignant pulmonary nodules, and may guide management of positive LDCT screening results. Additional clinical studies with larger sample size are needed to establish the robustness of this model.

X-chromosome inactivation in human iPSCs provides insight into X-regulated gene expression in autosomes

BackgroundVariation in X chromosome inactivation (XCI) in human-induced pluripotent stem cells (hiPSCs) can impact their ability to model biological sex biases. The gene-wise landscape of X chromosome gene dosage remains unresolved in female hiPSCs. To characterize patterns of de-repression and escape from inactivation, we performed a systematic survey of allele specific expression in 165 female hiPSC lines.ResultsXCI erosion is non-random and primarily affects genes that escape XCI in human tissues. Individual genes and cell lines vary in the frequency and degree of de-repression. Bi-allelic expression increases gradually after modest decrease of XIST in cultures, whose loss is commonly used to mark lines with eroded XCI. We identify three clusters of female lines at different stages of XCI. Increased XCI erosion amplifies female-biased expression at hypomethylated sites and regions normally occupied by repressive histone marks, lowering male-biased differences in the X chromosome. In autosomes, erosion modifies sex differences in a dose-dependent way. Male-biased genes are enriched for hypermethylated regions, and de-repression of XIST-bound autosomal genes in female lines attenuates normal male-biased gene expression in eroded lines. XCI erosion can compensate for a dominant loss of function effect in several disease genes.ConclusionsWe present a comprehensive view of X chromosome gene dosage in hiPSCs and implicate a direct mechanism for XCI erosion in regulating autosomal gene expression in trans. The uncommon and variable reactivation of X chromosome genes in female hiPSCs can provide insight into X chromosome’s role in regulating gene expression and sex differences in humans.

Hypermethylation of BMPR2 and TGF-β Promoter Regions in Tibetan Patients with High-Altitude Polycythemia at Extreme Altitude.

Although the expression of many genes is associated with adaptation to high-altitude hypoxic environments, the role of epigenetics in the response to this harsh environmental stress is currently unclear. We explored whether abnormal DNA promoter methylation levels of six genes, namely, ABCA1, SOD2, AKT1, VEGFR2, TGF-β, and BMPR2, affect the occurrence and development of high-altitude polycythemia (HAPC) in Tibetans. The methylation levels of HAPC and the control group of 130 Tibetans from very high altitudes (> 4500m) were examined using quantitative methylation-specific real-time PCR (QMSP). Depending on the type of data, the Pearson chi-square test, Wilcoxon rank-sum test, and Fisher exact test were used to assess the differences between the two groups. The correlation between the methylation levels of each gene and the hemoglobin content was explored using a linear mixed model. Our experiment revealed that the methylation levels of the TGF-β and BMPR2 genes differed significantly in the two groups (p < 0.05) and linear mixed model analysis showed that the correlation between the hemoglobin and methylation of ABCA1, TGF-β, and BMPR2 was statistically significant (p < 0.05). Our study suggests that levels of TGF-β and BMPR2 methylation are associated with the occurrence of HAPC in extreme-altitude Tibetan populations among 6 selected genes. Epigenetics may be involved in the pathogenesis of HAPC, and future experiments could combine gene and protein levels to verify the diagnostic value of TGF-β and BMPR2 methylation levels in HAPC.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Genetic and epigenetic alterations in precursor lesions of endometrial endometrioid carcinoma.

The hyperplasia-carcinoma sequence is a stepwise tumourigenic programme towards endometrial cancer in which normal endometrial epithelium becomes neoplastic through non-atypical endometrial hyperplasia (NAEH) and atypical endometrial hyperplasia (AEH), under the influence of unopposed oestrogen. NAEH and AEH are known to exhibit polyclonal and monoclonal cell growth, respectively; yet, aside from focal PTEN protein loss, the genetic and epigenetic alterations that occur during the cellular transition remain largely unknown. We sought to explore the potential molecular mechanisms that promote the NAEH-AEH transition and identify molecular markers that could help to differentiate between these two states. We conducted target-panel sequencing on the coding exons of 596 genes, including 96 endometrial cancer driver genes, and DNA methylome microarrays for 48 NAEH and 44 AEH lesions that were separately collected via macro- or micro-dissection from the endometrial tissues of 30 cases. Sequencing analyses revealed acquisition of the PTEN mutation and the clonal expansion of tumour cells in AEH samples. Further, across the transition, alterations to the DNA methylome were characterised by hypermethylation of promoter/enhancer regions and CpG islands, as well as hypo- and hyper-methylation of DNA-binding regions for transcription factors relevant to endometrial cell differentiation and/or tumourigenesis, including FOXA2, SOX17, and HAND2. The identified DNA methylation signature distinguishing NAEH and AEH lesions was reproducible in a validation cohort with modest discriminative capability. These findings not only support the concept that the transition from NAEH to AEH is an essential step within neoplastic cell transformation of endometrial epithelium but also provide deep insight into the molecular mechanism of the tumourigenic programme. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.