Methylation Status Of Genes Research Articles

Genome-wide analysis of DNA methylation and its relationship with serum homocysteine levels in patients with hypertension.

Homocysteine (Hcy) is an independent risk factor for cardiovascular diseases, and elevated plasma Hcy levels could aggravate vascular injury in hypertension. Hyperhomocysteinemia can change the methylation status of global DNA and specific genes. In the present study, we aim to examine the comprehensive influence of Hcy levels on DNA methylation status in patients with hypertension. Epigenome-wide methylation profiles of the peripheral leukocyte DNA of 218 patients with hypertension were analyzed using the Illumina Infinium Methylation EPIC BeadChip. Differentially methylated positions (DMPs) associated with serum Hcy levels were identified by mixed linear regression with the adjustment of potential confounders. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were conducted to determine the potential functions of the identified DMPs. The association between the methylation level of DMPs and carotid-femoral pulse wave velocity (Cf-PWV) was also analyzed. Five DMPs at cg13169662, cg03179312, cg21976560, cg25262698, and cg09433843 showed significant association with serum Hcy levels (false discovery rate-corrected P < 0.05). An additional six CpG sites met the threshold for suggestive significance ( P < 1 × 10 -6 ), among which three DMPs (cg25781123, cg26463106, and cg06679221) were annotated to THUMPD3 . Furthermore, the methylation levels of cg13169662 and cg25262698 (RPRD1A) were significantly associated with Cf-PWV. Our results suggest that Hcy could induce DNA methylation alteration in patients with hypertension. Further functional research is warranted to elucidate the concrete role of DMPs in hypertension.

Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers.

SMAD4 is a potent tumor suppressor. SMAD4 loss increases genomic instability and plays a critical role in the DNA damage response that leads to skin cancer development. We aimed to investigate SMAD4 methylation effects on mRNA and protein expression of SMAD4 in cancer and healthy tissues from patients with basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and basosquamous skin cancer (BSC). The study included 17 BCC, 24 cSCC and nine BSC patients. DNA and RNA were isolated from cancerous and healthy tissues following punch biopsy. Methylation-specific polymerase chain reaction (PCR) and real-time quantitative PCR methods were used to examine SMAD4 promoter methylation and SMAD4 mRNA levels, respectively. The percentage and intensity of staining of the SMAD4 protein were determined by immunohistochemistry. The percentage of SMAD4 methylation was increased in the patients with BCC (p = 0.007), cSCC (p = 0.004), and BSC (p = 0.018) compared to the healthy tissue. SMAD4 mRNA expression was decreased in the patients with BCC (p˂0.001), cSCC (p˂0.001), and BSC (p = 0.008). The staining characteristic of SMAD4 protein was negative in the cancer tissues of the patients with cSCC (p = 0.00). Lower SMAD4 mRNA levels were observed in the poorly differentiated cSCC patients (p = 0.001). The staining characteristics of the SMAD4 protein were related to age and chronic sun exposure. Hypermethylation of SMAD4 and reduced SMAD4 mRNA expression were found to play a role in the pathogenesis of BCC, cSCC, and BSC. A decrease in SMAD4 protein expression level was observed only in cSCC patients. This suggests that epigenetic alterations to the SMAD4 gene are associated with cSCC. The name of the trial register: SMAD4 Methylation and Expression Levels in Non-melanocytic Skin Cancers; SMAD4 Protein Positivity. The registration number: NCT04759261 ( https://clinicaltrials.gov/ct2/results?term=NCT04759261 ).

Methylation status of various gene loci in localized prostate cancer: Novel biomarkers for diagnostics and biochemical recurrence.

Oncologic outcomes for patients with localized prostate cancer (PCa) undergoing radical prostatectomy (RP) can vary widely. Hypermethylation of tumor-associated genes has potential as a novel diagnostic tool and predictive biomarker in PCa. We investigated the methylation status of tumor-associated genes in patients who underwent RP. Patients who underwent RP during 2004 to 2008 were matched retrospectively based on post-operative D'Amico risk stratification. Quantitative pyrosequencing was used to analyze methylation status of 10 gene loci in cancerous and adjacent benign tissue from histological specimen. Follow-up was performed according to EAU guideline recommendations. Statistical analyses were performed to correlate methylation levels in cancerous and benign tissue with risk profiles and biochemical recurrence (BCR). The cohort included 71 patients: 22 low-risk, 22 intermediate-risk, and 27 high-risk. Mean follow-up time was 74 months. Methylation status differed significantly between cancerous and adjacent benign tissue for the 5 gene loci GSTP1, APC, RASSF1, TNFRFS10c, and RUNX3 (each P < 0.001). Also, the methylation level was significantly higher in high-risk than in low-risk patients for Endoglin2 and APC (P = 0.026; P = 0.032). Using ROC analysis, hypermethylation of APC in PCa tissue was associated with higher risk of BCR (P = 0.005). Methylation status of various gene loci holds diagnostic and predictive potential in PCa. Hypermethylation of APC, RASSF1, TNFRFS10c and RUNX3 were identified as novel PCa-specific biomarkers. Furthermore, increased methylation levels of APC and Endoglin2 were associated with high-risk PCa. Additionally, hypermethylation of APC was associated with increased risk of BCR after RP.

Multi-Omics Data Analysis Identifies Prognostic Biomarkers across Cancers.

Combining omics data from different layers using integrative methods provides a better understanding of the biology of a complex disease such as cancer. The discovery of biomarkers related to cancer development or prognosis helps to find more effective treatment options. This study integrates multi-omics data of different cancer types with a network-based approach to explore common gene modules among different tumors by running community detection methods on the integrated network. The common modules were evaluated by several biological metrics adapted to cancer. Then, a new prognostic scoring method was developed by weighting mRNA expression, methylation, and mutation status of genes. The survival analysis pointed out statistically significant results for GNG11, CBX2, CDKN3, ARHGEF10, CLN8, SEC61G and PTDSS1 genes. The literature search reveals that the identified biomarkers are associated with the same or different types of cancers. Our method does not only identify known cancer-specific biomarker genes, but also proposes new potential biomarkers. Thus, this study provides a rationale for identifying new gene targets and expanding treatment options across cancer types.

Epigenetic Silencing of p16INK4a gene in Sporadic Breast Cancer.

Epigenetic alterations of tumor suppressor genes (TSG) involved in the onset and progression of Breast Cancer (BC) may serve as biomarkers for early detection and prediction of disease prognosis. We have herein tried to determine the methylation status of TSG, p16INK4a, in our 50 BC patients and their association with clinicopathological parameters. The methylation status of the p16INK4a gene in fresh tissue samples from 50 patients with BC was assessed by methylation-specific polymerase chain reaction (MS-PCR). The mean age of BC patients was 49.30 ± 9.75years. Of 50 BC samples tested, 21 (42%) had methylated p16INK4a gene. p16INK4a gene hypermethylation was significantly associated with age ≤ 50years, premenopausal status and advanced BC stage. Multivariate analysis revealed a strong association between advanced BC stage (Stage III and Stage IV) and p16INK4a hypermethylation (P = 0.008, RR = 5.996, 95% CI = 1.581-22.739). p16INK4a methylation was significantly associated with Triple Negative BC (TNBC) (P = 0.045, OR = 4.181, 95% CI = 1.030-16.981). These findings indicate that p16INK4a hypermethylation frequently occurs in BC. Hypermethylation of p16INK4a in young, premenopausal, TNBC and with advance stage in BC patients suggests its association with aggressive BC.

Investigation of the relationship between DNA methylation of PTH, ESRRA, FSHR and obesity: A single center study

Background: Obesity is a multifactorial disorder that is an important predisposing factor for a number of disorders. Genetic and epigenetic variations play important roles during the development of obesity. Method: DNA methylation is the most studied as one of the epigenetic modifications and is directly related to gene expression. Here, the methylation status of FSHR, PTH and ESRRA genes were investigated in obese patients to discover novel associations between DNA methylation and obesity phenotype. A total of 69 patients with obesity and 76 patients without obesity were enrolled in this study. DNA methylation in FSHR, PTH and ESRRA genes was analyzed. Result: There is a statistically significant association between the methylation status of ESRRA and fasting glucose (P=0.04) and BMI and methylation of the PTH gene (P=0.036) in obese subjects. In non-obese subjects, a statistically significant association was detected between adiponectin, resistin levels and FSHR methylation status. It is concluded that the methylation status of FSHR, PTH and ESRRA genes play an important role in obesity phenotype, adiponectin and resistin levels. These genes had previously been associated with obesity-related variables, but their methylation status was not highlighted. Conclusion: Our study will shed light on resolving the epigenetic contributions toward the development of obesity. Studies in the field of epigenetics will help discover predisposition markers for obesity. Based on the literature this was the first study that shows the interactions between FSHR, PTH and ESRRA methylation and obesity. Bangladesh Journal of Medical Science Vol. 22 No. 03 July’23 Page : 623-631

How Does Nutrition Affect the Epigenetic Changes in Dairy Cows?

Dairy cows require a balanced diet that provides enough nutrients to support milk production, growth, and reproduction. Inadequate nutrition can lead to metabolic disorders, impaired fertility, and reduced milk yield. Recent studies have shown that nutrition can affect epigenetic modifications in dairy cows, which can impact gene expression and affect the cows' health and productivity. One of the most important epigenetic modifications in dairy cows is DNA methylation, which involves the addition of a methyl group to the DNA molecule. Studies have shown that the methylation status of certain genes in dairy cows can be influenced by dietary factors such as the level of methionine, lysine, choline, and folate in the diet. Other important epigenetic modifications in dairy cows are histone modification and microRNAs as regulators of gene expression. Overall, these findings suggest that nutrition can have a significant impact on the epigenetic regulation of gene expression in dairy cows. By optimizing the diet of dairy cows, it may be possible to improve their health and productivity by promoting beneficial epigenetic modifications. This paper reviews the main nutrients that can cause epigenetic changes in dairy cattle by analyzing the effect of diet on milk production and its composition.

Gene body hypomethylation of pyroptosis-related genes NLRP7, NLRP2, and NLRP3 facilitate non-invasive surveillance of hepatocellular carcinoma.

Programmed cell death (PCD) resistance is a key driver of cancer occurrence and development. The prognostic relevance of PCD-related genes in hepatocellular carcinoma (HCC) has attracted considerable attention in recent years. However, there is still a lack of efforts to compare the methylation status of different types of PCD genes in HCC and their roles in its surveillance. The methylation status of genes related to pyroptosis, apoptosis, autophagy, necroptosis, ferroptosis, and cuproptosis was analyzed in tumor and non-tumor tissues from TCGA. Whole-genome bisulfite sequencing (WGBS) data of paired tumor tissue and buffy coat samples were used to filter the potential interference of blood leukocytes in cell-free DNA (cfDNA). The WGBS data of healthy individuals' and early-stage HCC patients' cfDNA were analyzed to evaluate the distinguishing ability. The average gene body methylation (gbDNAme) of pyroptosis-related genes (PRGs) was significantly altered in HCC tissues relative to normal tissues, and their distinguishing ability was higher compared to the other types of PCD-related genes. The gbDNAme of NLRP7, NLRP2, and NLRP3 was reflective of the hypomethylation in HCC tissues, and methylation levels of NLRP3 correlated positively with its expression level (r=0.51). The candidate hypomethylated PRGs could discriminate between early HCC patients and healthy controls in cfDNA analysis with high accuracy (area under the receiver operation curve, AUC=0.94). Furthermore, the hypomethylation of PRGs was associated with poor prognosis of HCC. Gene body hypomethylation of PRGs is a promising biomarker for early HCC detection, monitoring of tumor recurrence, and prognosis prediction.

Key genes involved with prognosis were identified in lung adenocarcinoma by integrated bioinformatics analysis

ObjectiveBy screening the core genes in lung adenocarcinoma (LUAD) with bioinformatics, our study evaluated its prognosis value and role in infiltration process of immune cells. MethodsUsing GEO database, we screened 5 gene chips, including GSE11072, GSE32863, GSE43458, GSE115002, and GSE116959. Then, we obtained the corresponding differentially expressed genes by analyzed 5 gene chips online by GEO2R (P < 0.05, |logFC| > 1). Then, through DAVID online platform, Cytoscape 3.6.1 software and PPI network analysis, the network was visualized and obtain the final core genes. Next, we plan to use the GEPIA, UALCAN, Kaplan–Meier plotter and Time 2.0 database for corresponding analysis. The GEPIA database was used to verify the expression of core genes in LUAD and normal lung tissues, and survival analysis was used to evaluate the value of core genes in the prognosis of LUAD patients. UALCAN was used to verify the expression of the LUAD core gene and promoter methylation status, and the predictive value of core genes was evaluated in LUAD patients by the Kaplan–Meier plotter online tool. Then, we used the Time 2.0 database to identify the relationship to immune infiltration in LUAD. Finally, we used the human protein atlas (HPA) database for online immunohistochemical analysis of the expressed proteins. ResultsThe expression of CCNB2 and CDC20 in LUAD were higher than those in normal lung tissues, their increased expression was negatively correlated with the overall survival rate of LUAD, and they were involved in cell cycle signal transduction, oocyte meiosis signal transduction as well as the infiltration process of immune cells in LUAD. The expression proteins of CCNB2 and CDC20 were also different in lung cancer tissue and normal lung tissue. Therefore, CCNB2 and CDC20 were identified as the vital core genes. ConclusionCCNB2 and CDC20 are essential genes that may constitute prognostic biomarkers in LUAD, they also participate the immune infiltration process and protein expression process of LUAD, and might provides basis for clinical anti-tumor drug research.

MiR-137–LAPTM4B regulates cytoskeleton organization and cancer metastasis via the RhoA-LIMK-Cofilin pathway in osteosarcoma

Osteosarcoma (OS) is a rare malignant bone tumor but is one leading cause of cancer mortality in childhood and adolescence. Cancer metastasis accounts for the primary reason for treatment failure in OS patients. The dynamic organization of the cytoskeleton is fundamental for cell motility, migration, and cancer metastasis. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is an oncogene participating in various biological progress central to cancer biogenesis. However, the potential roles of LAPTM4B in OS and the related mechanisms remain unknown. Here, we established the elevated LAPTM4B expression in OS, and it is essential in regulating stress fiber organization through RhoA–LIMK–cofilin signaling pathway. In terms of mechanism, our data revealed that LAPTM4B promotes RhoA protein stability by suppressing the ubiquitin-mediated proteasome degradation pathway. Moreover, our data show that miR-137, rather than gene copy number and methylation status, contributes to the upregulation of LAPTM4B in OS. We report that miR-137 is capable of regulating stress fiber arrangement, OS cell migration, and metastasis via targeting LAPTM4B. Combining results from cells, patients’ tissue samples, the animal model, and cancer databases, this study further suggests that the miR-137–LAPTM4B axis represents a clinically relevant pathway in OS progression and a viable target for novel therapeutics.

Different resistance exercise loading paradigms similarly affect methylation status and mRNA expression patterns of myostatin-related genes in skeletal muscle

Although several resistance exercise studies have used bioinformatics platforms to identify the biological relevance of gene expression changes, these platforms seldom provide in-depth information on genes that have been mechanistically linked to skeletal muscle hypertrophy. Thus, we sought to perform a secondary analysis on a muscle transcriptomic dataset we previously collected involving two different bouts of resistance exercise. Previously trained college-aged males (n=11, training experience: 4±3 years) performed two resistance exercise bouts separated by one week. The higher-load bout (80fail) consisted of 4 sets of back squats and 4 sets of leg extensions to failure using 80% of their estimated one-repetition maximum. The lower-load bout (30fail) consisted of this same paradigm using 30% of their one-repetition maximum. Vastus lateralis muscle biopsies were collected before (PRE), 3 hours (3h), and 6 hours (6h) after each exercise bout, and bouts were separated by a one-week washout. Muscle mRNA was analyzed for genome-wide mRNA expression patterns using the Clariom S mRNA array. Based on an extensive literature search performed by us and others, the mRNA expression profile of gene candidates mechanistically associated with skeletal muscle hypertrophy (58 genes) was interrogated between 30fail and 80fail training. Select targets were further interrogated for associated protein expression and phospho-signaling events. Although none of the 58 skeletal muscle hypertrophy-associated gene targets demonstrated significant bout✕time point interactions, ~57% showed a significant time-only effect from PRE to 3h (15↑ and 18↓, p&lt;0.01) and ~26% showed significant time-only effect from PRE to 6h (8↑ and 9↓, p&lt;0.01). Genes related to myostatin signaling (9 genes) and mTORC1 signaling (9 genes) were well represented on the gene list. Compared to mTORC1 signaling mRNAs, a greater percentage of myostatin signaling-related mRNAs were dynamically altered post-exercise (i.e., significant time-only effects). There were increases in phospho (Thr389)/pan p70S6K (p=0.001; PRE to 3h) and follistatin protein levels (p=0.021; PRE to 6h) regardless of bout. No significant time effects or interactions were observed for phospho (Ser2448)/pan mTOR, phospho (Thr389)/pan AKT, phospho (Ser235/236)/pan rpS6, or myostatin protein levels. The data have multiple implications. First, performing lighter load and heavier load resistance exercise to failure elicits similar alterations in the mRNA responses of genes mechanistically associated with skeletal muscle hypertrophy and mTORC1 signaling. Second, acute resistance exercise predominantly affects the myostatin signaling pathway via transcriptional mechanisms and genes involved with mTORC1 signaling are less affected in this regard. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The relationship of promoter methylation of calcium voltage-gated channel alpha 1 and interleukin-16 to primary osteoarthritis.

Osteoarthritis is the most prevalent joint disease worldwide and the primary cause of musculoskeletal dysfunction. Epigenetic changes in various genes, particularly methylation, have been implicated as possible underlying causes of primary osteoarthritis. The aim of our study was to investigate the promoter methylation status of the calcium voltage-gated channel alpha 1 subunit G (CACNA1G) and interleukin-16 (IL-16) genes, which are strongly associated with calcium channel activity and antigen presentation, respectively, in primary osteoarthritis patients. Twenty-one patients with primary osteoarthritis and 25 healthy controls were included in our study. The methylation status of CACNA1G and IL-16 genes was analyzed with methylation-specific Polymerase Chain Reaction (PCR), and the serum levels of IL-16 were determined with Enzyme-Linked Immunosorbent Assay (ELISA). The age of the patients was 63.95±8.41 years, and they were 15 females and 6 males. The promoter of the CACNA1G gene was found to be hypermethylated in primary osteoarthritis patients (p<0.001). In contrast, the promoter of the IL-16 gene was found to be hypomethylated compared to the control (p<0.001). The serum levels of IL-16 increased in parallel with the hypomethylated promoter status of IL-16 gene in primary osteoarthritis patients compared to the control (p<0.001). Our study indicates that the methylation status of CACNA1G and IL-16 gene promoters are epigenetically altered in patients with primary osteoarthritis. Moreover, increased serum IL-16 levels in osteoarthritis patients may be associated with hypomethylation of the IL-16 gene promoter.

Promoter Methylation of Two HOXA9 and NISCH Genes in Opium Users.

Opiate abuse has been critically increased in the world, especially in Iran. Owing to the association of opiate use with multiple human cancers and neurological disorders, seeking for genetic and epigenetic effects of opium can pave the way for early diagnosis of major health defects in addicted users. Accordingly, the present study aimed to determine the methylation status of the promoter of two genes, which are actively involved in neurodevelopment and cancer evolution. DNA was isolated from peripheral blood of 28 opium abusers and 19 healthy controls and then subjected to sonication. Sonicated DNAs undergone methylated DNA immunoprecipitation-real time polymerase chain reaction (MeDIP-Real Time PCR) using specific primer pairs designed for HOXA9 and NISCH genes. Obtained data were analyzed using SPSS software. HOXA9 and NISCH genes were found to be significantly methylated in addicted users compared to controls (P<0.001) which was significantly associated with the mean of the age regarding HOXA9 gene (P=0.002). Neither opium amount nor duration or route of using was associated with the methylation status of HOXA9 or NISCH genes. Hypermethylation of HOXA9 and NISCH genes as tumor suppressor in opium-addicted individuals can be considered as confirmatory evidence for carcinogenesis of opium. Further studies are required to figure out the role of epigenetic alterations in cancer evolution among opium users.

P129 DNA methylation of the tumour necrosis factor gene as a biomarker of diagnosis in early rheumatoid arthritis patients

Abstract Background/Aims Rheumatoid arthritis (RA) is an autoimmune inflammatory condition that affects the synovial joints. RA exists on a disease continuum where immunological changes occur before the onset of clinical symptoms. Changes in DNA methylation status of genes are thought to be early triggers of the progression from health to disease. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that contributes to RA. Recently it has been shown that the loss of DNA methylation in the promoter region of the TNF gene is related to early RA. This study aims to establish the predictive value of the levels of DNA methylation of the TNF gene as a biomarker for diagnosis of RA. Methods Blood samples from a retrospective cohort of patients with symptoms of inflammatory arthritis between 2010 and 2019 at the Leeds Early Arthritis Clinic were selected. Data obtained include clinical symptoms of disease progression over 2 years to establish a diagnosis. DNA methylation levels of the TNF gene were measured by quantitative polymerase chain reaction (qPCR) and analysed using binary logistic regression with forward (LR) variable selection for added value as a diagnostic biomarker. Results For the 284 patients with a mean age of 53.8, 202 (71.7%) were female and 82 (28.9%) were male. From N = 284, 190 (66.9%) were diagnosed with RA and 94 (33.1%) with another conditions. The predictive accuracy of the regression model was increased by 3.5% from 85.8% to 89.3% when TNF DNA-methylation levels was added to demographic and clinical variables. The AUC of the model with TNF was 0.940 compared to 0.911 without TNF DNA-methylation levels. The sensitivity and specificity of the prediction with DNA methylation data were 94.6% and 77.9% respectively. This TNF model has a positive predictive value of 90.2% and a negative predictive value of 87.0%. Conclusion DNA methylation of the TNF gene is an important biomarker for RA diagnosis. This study showed that including levels of TNF DNA-methylation improves the accuracy of diagnosing RA. Disclosure M.C. Liu: None. H. Ng: None. F. Ponchel: None.

Abstract 6023: Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer

Abstract Background: DNA silencing of gene expression can affect tumorigenesis, treatment outcomes and prognosis. Epigenetic silencing of tumor suppressor genes in prostate cancer detected in plasma at different stages of progression and its effect on clinical outcomes has not been determined. In a cohort study across different stages of prostate cancer (PC) progression, we performed plasma cell-free DNA (cfDNA)-based epigenomic profiling for determining the landscape of methylation and Differentially Methylated Region (DMR) classifiers and the potential impact on clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) state. Methods: Plasma was collected prospectively in a longitudinal cohort study of 108 PC patients (pts) of which 24 had non-metastatic Local PC; 28 had mHSPC; 56 had mCRPC. cfDNA was used for enzymatic methylation sequencing (EM-seq) of a targeted panel of genes (N=441) implicated in prostate cancer biology. Methylation status at CpG islands of these genes were examined using Twist targeted cfDNA methylation assay. The Bismark program was used for methylation calls. Temporal trends of differentially methylated regions (DMRs) were identified in different states of progression by comparing the methylation status of target genes in localized PC, mHSPC and mCRPC states using a False Discovery Rate (FDR-q-value) of 0.2 or less. DMRs uniquely associated with mHSPC state were identified and determination of genes with DMRs in pts experiencing early failure of androgen deprivation therapy (ADT)-based therapies was performed. Early failure was defined as progression on ADT within 12 months. Results: We identified 304/441 genes that were differentially methylated between localized PC and advanced PC (mHSPC and mCRPC) (FDR&amp;lt;0.2), of which 237 genes differed between mHSPC and mCRPC (p&amp;lt;0.05) states. mHSPC pts were serially profiles before and after 3 months of ADT-based therapies. All 28 pts experienced a PSA response at 3-months and 112/237 genes showed significant change in methylation status in these serial samples taken before/after 3 months of ADT-based therapies. The median follow up of the cohort was 18 months (range 10-24). 6/28 mHSPC pts experienced early ADT failure (&amp;lt;12 months; median 10 months). 8/112 genes were significantly associated with early failures and included DMRs in RB1, FOXA1, SOX11. GSC, ALDH1L1, SCGB3A1, FLT4, and PTPRN2. Conclusions. A targeted cfDNA plasma liquid-biopsy based epigenomic profiling of PC cancer related genes was able to capture significant differences in DMRs across different states of cancer progression. Candidate differentially expressed CpG methylome gene biomarkers detected in plasma also identified biomarkers associated with early ADT-based treatment failure in mHSPC. Citation Format: Manish Kohli, Jodie Wong, Yijun Tian, Amir Bitaraf, Claire Hanson, Matt Larsen, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Jonathan Tward. Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer [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 6023.

Association of DNA Promoter Methylation and BRAF Mutation in Thyroid Cancer.

The BRAF V600E mutation and DNA promoter methylation play important roles in the pathogenesis of thyroid cancer (TC). However, the association of these genetic and epigenetic alterations is not clear. In this study, using paired tumor and surrounding normal tissue from the same patients, on a genome-wide scale we tried to identify (a) any association between BRAF mutation and DNA promoter methylation, and (b) if the molecular findings may provide a basis for therapeutic intervention. We included 40 patients with TC (female = 28, male = 12) without distant metastasis. BRAF mutation was present in 18 cases. We identified groups of differentially methylated loci (DML) that are found in (a) both BRAF mutant and wild type, (b) only in BRAF mutant tumors, and (c) only in BRAF wild type. BRAF mutation-specific promoter loci were more frequently hypomethylated, whereas BRAF wild-type-specific loci were more frequently hypermethylated. Common DML were enriched in cancer-related pathways, including the mismatch repair pathway and Wnt-signaling pathway. Wild-type-specific DML were enriched in RAS signaling. Methylation status of checkpoint signaling genes, as well as the T-cell inflamed genes, indicated an opportunity for the potential use of PDL1 inhibitors in BRAF mutant TC. Our study shows an association between BRAF mutation and methylation in TC that may have biological significance.

Targeted epigenetic silencing of UCHL1 expression suppresses collagen-1 production in human lung epithelial cells

ABSTRACT Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is highly expressed in smokers, but little is known about the molecular mechanism of UCHL1 in airway epithelium and its possible role in affecting extracellular matrix (ECM) remodelling in the underlying submucosa. Since cigarette smoking is a major cause of lung diseases, we studied its effect on UCHL1 expression and DNA methylation patterns in human bronchial epithelial cells, obtained after laser capture micro-dissection (LCM) or isolated from residual tracheal/main stem bronchial tissue. Targeted regulation of UCHL1 expression via CRISPR/dCas9 based-epigenetic editing was used to explore the function of UCHL1 in lung epithelium. Our results show that cigarette smoke extract (CSE) stimulated the expression of UCHL1 in vitro. The methylation status of the UCHL1 gene was negatively associated with UCHL1 transcription in LCM-obtained airway epithelium at specific sites. Treatment with a UCHL1 inhibitor showed that the TGF-β1-induced upregulation of the ECM gene COL1A1 can be prevented by the inhibition of UCHL1 activity in cell lines. Furthermore, upon downregulation of UCHL1 by epigenetic editing using CRISPR/dCas-EZH2, mRNA expression of COL1A1 and fibronectin was reduced. In conclusion, we confirmed higher UCHL1 expression in current smokers compared to non- and ex-smokers, and induced downregulation of UCHL1 by epigenetic editing. The subsequent repression of genes encoding ECM proteins suggest a role for UCHL1 as a therapeutic target in fibrosis-related disease.

Regulation of Shh/Bmp4 Signaling Pathway by DNA Methylation in Rectal Nervous System Development of Fetal Rats with Anorectal Malformation

ObjectiveTo study the influence of gene methylation in the Shh/Bmp4 signaling pathway on the enteric nervous system in the rectum of rat embryos with anorectal malformations (ARMs). MethodsPregnant Sprague Dawley rats were divided into three groups; two groups treated with either ethylene thiourea (ETU induce ARM) or ETU+5-azacitidine (5-azaC inhibit DNA methylation) and a normal control group. The levels of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b), the methylation status of the Shh gene promoter region and the expression of the key components were detected by PCR, immunohistochemistry and western blotting. ResultsThe expression of DNMTs in the rectal tissue of the ETU and ETU+5-azaC groups was higher than that of the control. The expression of DNMT1, DNMT3a and methylation level of the Shh gene promoter in the ETU group was higher than in the ETU+5-azaC group (P < 0.01). The methylation level of the Shh gene promoter was higher in the ETU+5-azaC group than in the control. The Shh and Bmp4 expression in the ETU and ETU+5-azaC groups were lower than in the control, and their expression in the ETU group was also lower than in the ETU+5-azaC group. ConclusionThe methylation status of genes in the rectum of the ARM rat model may be changed by intervention. The low methylation level of the Shh gene may promote the expression of key Shh/Bmp4 signaling pathway components.

Value of Methylation Status of RPRM, SDC2, and TCF4 Genes in Plasma for Gastric Adenocarcinoma Screening.

To explore the clinical value of the combined screening of the methylation statuses of the RPRM, SDC2, and TCF4 genes in plasma of gastric cancer patients. Differential expressed genes (DEGs) were selected from the Gene Expression Omnibus database, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using DAVID, and a protein-protein interaction network was constructed. Hub genes were obtained using Cytoscape. Screening results combined with literature reports identified three genes (RPRM, SDC2, and TCF4). Further analysis was done using biopsies collected through gastroscopy at Shanxi Cancer Hospital from January 8, 2020 to February 22, 2021. The patients were divided into two groups: gastric adenocarcinoma group, and control group which are not gastric adenocarcinoma according to pathological or gastroscopic results. The methylation statuses of the three genes in peripheral blood plasma were detected by fluorescence polymerase chain reaction, and the relationships between the positive rates of the three combined genes with pathology and/or gastroscopy results were analyzed. The clinical value of the combined detection of the three genes was evaluated according to these indicators. The diagnostic specificity and sensitivity of this detective method were analyzed. A total of 197 DEGs were identified and 12 hub genes were obtained. The enriched functions and pathways of DEGs include regulation of cell proliferation, extracellular space, cytokine activity, and pathways in cancer. The combination of RPRM, SDC2, and TCF4 gene methylation had a specificity of 93.39% and sensitivity of 80.33%. The combined positive rate of RPRM, SDC2, and TCF4 gene methylation in patients with gastric adenocarcinoma was significantly higher compared with those without gastric adenocarcinoma (χ2=151.179, P<0.05). Combined detection of RPRM, SDC2, and TCF4 gene methylation in peripheral blood plasma maybe helpful in screening for gastric adenocarcinoma, and maybe a complementary method to gastroscopy and serum tumor markers.

Methylation status of potential genes in breast cancer patients and correlate them with gene expression

Breast cancer is one of the world’s most prevalent cancer among women. Globally approximately 2.3 million women are diagnosed with breast cancer in 2020. It arises due to epigenetic modification which outlooks aberrant methylation as its major cause. The methylation of DNA involves a covalent chemical modification which is one of the major drawback, promoting development of breast cancer. In addition, evidence and investigation suggest that these methylations at promoter region of CpG islands leads to silencing of gene associated with tumor suppressor which is involved in crucial gene expression leading to establishment of breast cancer. Among variety of hyper methylated gene involved in different function linked with breast cancer, the hyper methylation of BRCA1 gene is identified as the targeted gene in precipitating disorder. A lot of susceptible genes are identified and based on their penetrance ability are being classified In order to analyze DNA methylation status Bisulfite sequencing is employed which is based on the mechanism of converting every un-methylated cytosine into uracil. With the advancement in gene sequencing the concept of whole genome bisulfite sequencing is also on the way to determine the DNA methylation occurring at single base. Another robust and sensitive approach to determine the methylated status is RT-PCR, a diagnostic tool applied in order to detect the expression of single gene