Expression Of DNA Methyltransferase Research Articles

Shema Oral Liquid Ameliorates the Severity of LPS-Induced COPD via Regulating DNMT1.

Background: Chronic obstructive pulmonary disease (COPD) is the most common respiratory disease with high morbidity and mortality. Shema oral liquid (Shema) is a traditional Chinese medicine (TCM) approved for the treatment of respiratory diseases. Clinical applications have shown that Shema has antitussive, expectorant, and anti-asthmatic effects, but its definite efficacy to COPD is still unclear. This study aimed to explore the therapeutic capacity and potential mechanism of Shema in treatment of COPD. Methods: Network pharmacology was used to investigated the possible pharmacological mechanism of Shema against COPD. A rat model of lipopolysaccharide (LPS)-induced COPD was established to determine pulmonary ventilatory function, serum inflammatory cytokines, and pulmonary pathological change. Subsequently, tandem mass tag (TMT)-based quantitative proteomics was used to further reveal the therapeutic targets related with Shema against COPD. Western blot was finally performed to validate the expression of targeted proteins screened by proteomics research. Results: Network pharmacology analysis indicated that Shema against COPD mainly inhibited the inflammation and affected the immune system. The animal experiment demonstrated that Shema treatment protected the lung tissue from LPS induced injury, inhibited the levels of serum inflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α, and improved the respiratory ventilatory function by upregulating forced expiratory volume in 0.1 s (FEV0.1), FEV0.3, forced vital capacity (FVC), and the ratios of FEV0.1 (0.3)/FVC. Proteomic analysis and western blot both proved that Shema inhibited the expression of DNA methyltransferase 1 (DNMT1) in the lung tissue. Conclusion: The therapeutic mechanism of Shema in treatment of COPD may involve inhibiting inflammatory response, improving pulmonary ventilatory function, and alleviating LPS-induced lung injury through regulating the expression of DNMT1. This study also shed light on the development of therapeutic strategies in treating COPD by intervening DNMT-related pathways.

CircPCNX Promotes PDGF-BB-Induced Proliferation and Migration of Human Aortic Vascular Smooth Muscle Cells Through Regulating miR-1278/DNMT1 Axis.

Human aortic vascular smooth muscle cells (HA-VSMCs) play vital roles in the pathogenesis of vascular diseases. Circular RNAs (circRNAs) have been reported to regulate the biological functions of HA-VSMCs. In this study, the functions of circRNA pecanex homolog (circPCNX) in platelet-derived growth factor-BB (PDGF-BB)-induced HA-VSMCs were investigated. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine the expression of circPCNX, DNA methyltransferase 1 (DNMT1), and microRNA-1278 (miR-1278). 5'-Ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry analysis, wound healing assay, and transwell assay were used to examine cell proliferation, cell cycle, and migration. Western blot assay was utilized to measure protein levels. RNA immunoprecipitation (RIP) assay, RNA pull down assay, and dual-luciferase reporter assay were adopted to analyze the relationships among circPCNX, miR-1278, and DNMT1. CircPCNX was upregulated in PDGF-BB-treated HA-VSMCs in a dose- or time-dependent manner. CircPCNX knockdown alleviated PDGF-BB-induced cell proliferation, cell cycle progression, and migration in HA-VSMCs. CircPCNX knockdown could reverse PDGF-BB-induced HA-VSMC progression by regulating DNMT1. Moreover, circPCNX was identified to regulate DNMT1 expression by sponging miR-1278. Inhibition of miR-1278 reversed circPCNX knockdown-mediated effects on cell proliferation and migration in PDGF-BB-induced HA-VSMCs. MiR-1278 overexpression suppressed PDGF-BB-stimulated HA-VSMC proliferation and migration by targeting DNMT1. CircPCNX promoted PDGF-BB-induced HA-VSMC proliferation and migration by elevating DNMT1 expression through sponging miR-1278.

High DNMT1 Expression in Stromal Fibroblasts Promotes Angiogenesis and Unfavorable Outcome in Locally Advanced Breast Cancer Patients

BackgroundActive breast cancer-associated fibroblasts (CAFs) play a leading role in breast carcinogenesis through promoting angiogenesis and resistance to therapy. Consequently, these active stromal cells have significant influence on patient outcome. Therefore, we explored here the role of the DNA methyltransferase 1 (DNMT1) protein in CAF-dependent promotion of angiogenesis as well as the prognostic power of DNMT1 level in both cancer cells and their adjacent CAFs in locally advanced breast cancer patients.MethodsWe applied immunohistochemistry to evaluate the level of DNMT1 in breast cancer tissues and their adjacent normal counterparts. Quantitative RT-PCR and immunoblotting were performed to investigate the role of DNMT1 in regulating the expression of pro-angiogenic genes in active CAFs and also their response to the DNMT1 inhibitors decitabine (DAC) as well as eugenol.ResultsWe have shown that DNMT1 controls the pro-angiogenic potential of CAFs both in vitro and in vivo through positive regulation of the expression/secretion of 2 important pro-angiogenic factors VEGF-A and IL-8 as well as their upstream effectors mTOR and HIF-1α. To confirm this, we have shown that these DNMT1-related pro-angiogenic effects were suppressed by 2 DNMT1 inhibitors decitabine and eugenol. Interestingly, in a cohort of 100 tumors from locally advanced breast cancer patients (LABC), we have shown that high expression of DNMT1 in tumor cells and their adjacent stromal fibroblasts is correlated with poor survival of these patients.ConclusionDNMT1 upregulation in breast stromal fibroblasts promotes angiogenesis via IL-8/VEGF-A upregulation, and correlates well with poor survival of LABC patients.

Regulation of the cellular redox state and the expression of DNA methyltransferase-1 in peripheral blood mononuclear cells from patients with Graves’ disease

BackgroundGraves’ disease is an autoimmune disorder characterised by excessive production of thyroid hormones, which induces increased cellular metabolism in most tissues and increased production of reactive oxygen species (ROS). The aim of this work was to analyse the effect of ROS on cell viability and the expression of catalase (CAT), glutathione peroxidase-1 (GPx-1), superoxide dismutase (SOD-1) and DNA methyltransferase-1 (DNMT-1) in peripheral blood mononuclear cells (PBMC) from patients with newly diagnosed Graves’ disease or treated with methimazole. Patients and methodsFor this study, women patients with newly diagnosed Graves’ disease (n=18), treated with methimazole (n=6) and healthy subjects (n=15) were recruited. ROS were evaluated by flow cytometry, and the viability/apoptosis of PBMC was analysed by flow cytometry and fluorescence microscopy. Genomic expression of CAT, GPx-1, SOD-1 and DNMT-1 was quantified by real-time PCR. ResultsWe found high levels of ROS and increased expression of CAT, GPx-1, SOD-1 and DNMT-1 in PBMC from patients with newly diagnosed Graves’ disease. Methimazole treatment reversed these parameters. Cell viability was similar in all study groups. ConclusionsROS induces the expression of CAT, GPx-1, and SOD-1. The activity of these enzymes may contribute to the protection of PBMC from the harmful effect of free radicals on cell viability. Increased expression of DNMT-1 may be associated with aberrant methylation patterns in immunoregulatory genes contributing to autoimmunity in Graves’ disease.

Comparative transcriptome analysis of heat-induced domesticated zebrafish during gonadal differentiation

BackgroundThe influence of environmental factors, especially temperature, on sex ratio is of great significance to elucidate the mechanism of sex determination. However, the molecular mechanisms by which temperature affects sex determination remains unclear, although a few candidate genes have been found to play a role in the process. In this study, we conducted transcriptome analysis of the effects induced by high temperature on zebrafish during gonad differentiation period.ResultsTotals of 1171, 1022 and 2921 differentially expressed genes (DEGs) between high temperature and normal temperature were identified at 35, 45 and 60 days post-fertilization (dpf) respectively, revealing that heat shock proteins (HSPs) and DNA methyltransferases (DNMTs) were involved in the heat-exposed sex reversal. The Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway that were enriched in individuals after heat treatment included Fanconi anemia (FA) pathway, cell cycle, oocyte meiosis and homologous recombination.ConclusionsOur study provides the results of comparative transcriptome analyses between high temperature and normal temperature, and reveals that the molecular mechanism of heat-induced masculinization in zebrafish is strongly related to the expression of HSPs and DNMTs and FA pathway during gonad differentiation.

Estradiol-mediated inhibition of DNMT1 decreases p53 expression to induce M2-macrophage polarization in lung cancer progression

Previous studies indicate that estrogen positively regulates lung cancer progression. Understanding the reasons will be beneficial for treating women with lung cancer in the future. In this study, we found that tumor formation was more significant in female EGFRL858R mice than in male mice. P53 expression levels were downregulated in the estradiol (E2)-treated lung cancer cells, female mice with EGFRL858R-induced lung cancer mice, and premenopausal women with lung cancer. E2 increased DNA methyltransferase 1 (DNMT1) expression to enhance methylation in the TP53 promoter, which led to the downregulation of p53. Overexpression of GFP-p53 decreased DNMT1 expression in lung cancer cells. TP53 knockout in mice with EGFRL858R-induced lung cancer not only changed gene expression in cancer cells but also increased the polarization of M2 macrophages by increasing C–C motif chemokine ligand 5 (CCL5) expression and decreasing growth differentiation factor 15 (GDF15) expression. The TP53 mutation rate was increased in females with late-stage but not early-stage lung cancer compared to males with lung cancer. In conclusion, E2-induced DNMT1 and p53 expression were negatively regulated each other in females with lung cancer, which not only affected cancer cells but also modulated the tumor-associated microenvironment, ultimately leading to a poor prognosis.

Global DNA hypomethylation and the expression profile of DNA methyltransferase genes in late-onset neonatal sepsis.

Aim: Infectious organisms tend to cause DNA methylation changes. Thus, this paper aims to study global DNA methylation and the expression of DNA methyltransferase (DNMT)genes in late-onset neonatal sepsis (LONS). Methods: Global and Alu DNA methylation and expression levels of DNMT were performed using 5mc ELISA, methylation-specific PCR and quantitative real-time-PCR, respectively for LONS and controls. Results: Significant hypomethylation of global DNA and Alu DNA methylation and lower expression of DNMT1 and DNMT3a were observed in LONS compared with controls. Receiver operating characteristic analysis of global and Alu DNA methylation showed good discrimination for the identification of LONS. Conclusion: The hypomethylation of global DNA and Alu elements is evident in neonates with LONS. This may be clinically useful for the prognosis of LONS.

Expression of genes involved in epigenetic modifications in patients with rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, and evidence revealed that environmental and genetic factors are involved in RA pathogenesis. This study aimed to explore the balance between genes involved in DNA methylation and demethylation in the peripheral blood of patients with RA. MethodsIn this cross-sectional study, ninety RA patients and ninety-two age and sex-matched normal subjects (NS) were enrolled, and RA was confirmed by an expert rheumatologist based on clinical and laboratory data. In addition, the Rheumatoid arthritis severity scale (RASS) and disease activity score (DAS-28) were measured. According to the disease severity, RA patients were categorized into three groups (mild, moderate, and severe). Peripheral blood was collected from subjects, and the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1), TET2, and TET3, as well as DNA methyltransferase 1 (DNMT1) genes, were evaluated using real-time polymerase chain reaction (RT-PCR) technique. Serum level of anti-cyclic citrullinated peptide (anti-CCP) antibody was measured by enzyme-linked immunosorbent assay (ELISA) technique. ResultsThis investigation showed that gene expression of TET1 and TET2 was significantly decreased in RA patients compared to NS. Gene expression of DNMT1 and TET3 was also down-regulated in RA patients compared to NS; however, the changes were not statistically significant. Moreover, the correlation matrix test demonstrated no significant association between some demographic and clinical data of RA patients with the expression of studied genes. ConclusionAccording to the findings of this study, the expression of TET1 and TET2 as regulators of inflammatory responses was remarkably reduced in RA patients compared to NS, and disease severity can affect the expression of these genes because the expression of the TET2 is significantly reduced in patients with moderate RA severity and it may lead to increase inflammatory responses and RA development.

Matrine inhibits advanced glycation end products-induced macrophage M1 polarization by reducing DNMT3a/b-mediated DNA methylation of GPX1 promoter

Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes-exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms. Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation. Matrine reduced AGEs exposure-elevated expressions of DNA methyltransferase (DNA MTase, DNMT)3a and DNMT3b in macrophages which were not affected by RAGE or TLR4 over expressions. DNA methylation rate of GPX1 promoter was reduced from 97.22% to 66.67% in AGEs- exposed macrophages treated by matrine. GPX1 expression was up-regulated by matrine, which further suppressed AGEs/RAGE-mediated oxidative stress. Thus, the activation of down-stream TLR4/STAT1 signaling pathway was inhibited by matrine treatment which eventually suppressed AGEs- induced macrophage M1 polarization. However, these effects of matrine were impaired by RAGE and TLR4 overexpression. Results from this study suggested that matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b-induced GPX1 promoter DNA methylation.

Joint Effects of Cigarette Smoking and Green Tea Consumption with miR-29b and DNMT3B mRNA Expression in the Development of Lung Cancer.

In tumor development, increased expression of DNA methyltransferase (DNMT) has been observed. In particular, cigarette smoke and tea polyphenols may influence DNMT3B mRNA expression by regulating microRNA (miR)-29b expression. Herein, we designed a case–control study to evaluate the joint effects of smoking and green tea consumption, with miR-29b and DNMT3B mRNA expression, in lung cancer development. A total of 132 lung cancer patients and 132 healthy controls were recruited to measure miR-29b and DNMT3B mRNA expression in whole blood. Results revealed that lung cancer patients had lower miR-29b expression (57.2 vs. 81.6; p = 0.02) and higher DNMT3B mRNA expression (37.2 vs. 25.8; p < 0.001) than healthy controls. Compared to non-smokers with both higher miR-29b and lower DNMT3B mRNA expression, smokers with both low miR-29b and higher DNMT3B mRNA expression had an elevated risk of lung cancer development (OR 5.12, 95% CI 2.64–9.91). Interactions of smoking with miR-29b or DNMT3B mRNA expression in lung cancer were significant. Interaction of green tea consumption with miR-29b expression and DNMT3B mRNA expression in lung cancer was also significant. Our study suggests that smokers and green tea nondrinkers with lower miR-29b expression and higher DNMT3B mRNA expression are more susceptible to lung cancer development.

Pan-Cancer Analyses Reveal Oncogenic and Immunological Role of PLOD2.

Some previous studies have shown that PLOD2 has some value in tumorigenesis. However, the broad significance of PLOD2 has not been discussed in depth. This study was aimed at elaborated and summarized the value of PLOD2 in various tumors. First, we integrated GTEx, The Cancer Genome Atlas and Cancer Cell Line Encyclopedia databases to analyze the expression of PLOD2, and found that it was expressed differently in normal tissues and significantly highly expressed in most tumors compared with normal tissues. Second, our analysis revealed that PLOD2 expression was negatively correlated with the prognosis of several tumors. For gastric cancer, the median overall survival time was significantly higher in the PLOD2 low expression group [HR 0.616 (95%CI 0.442–0.858), p = 0.004]. Third, for tumor immunity, PLOD2 was significantly associated with tumor infiltration, including immune infiltrating cells; immune checkpoint expression; immune microenvironment scores (immune score, stromal score and estimate scores); immunotherapy-related scores (tumor mutational burden, microsatellite instability, tumor neoantigen burden); expression of DNA repair genes Mismatch Repairs and methyltransferase; and enrichment analyses identified PLOD2-associated terms and pathways. Lastly, twenty pairs of gastric cancer and adjacent immunohistochemistry showed that PLOD2 was significantly overexpressed in gastric cancer (p < 0.001). Collectively, PLOD2 played a significant role in tumorigenesis and maybe serve as a potential biomarker for diagnosis and prognosis in cancers.

Differences in the expression of DNA methyltransferases and demethylases in leukocytes and the severity of pulmonary arterial hypertension between ethnic groups.

The loss of ten‐eleven translocation (TET2) methylcytosine dioxygenase expression contributes to the pathobiology of pulmonary arterial hypertension (PAH). However, whether the expression and activity of other TETs and DNA methyltransferases (DNMTs) are altered in PAH remains enigmatic. Therefore, our objective was to determine the expression of DNMT (1, 3a, and 3b) and TET (1, 2, and 3) and their total activity. We assessed the expression of DNMT and TET enzymes in the leukocytes and their activity in extracellular vesicles (EVs). Expression of DNMT (1, 3a, and 3b), TET (2 and 3) in leukocytes, and total activity in EVs, from PAH patients was higher than in healthy controls. Additionally, we noticed there were difference in expression of these epigenetic enzyme based on ethnicity and found higher DNMT1 and lower TET2/TET3 expression in Caucasian than Hispanic/African American (combine) patients. Since loss‐of‐function mutation(s) and down‐regulation of TET enzymes are associated with hematological malignancies and cytokine production, we determined the expression of genes that encode cytokines in samples of Caucasian and Hispanic/African American patients. Expression of IL6, CSF2, and CCL5 genes were higher in the leukocytes of Caucasian than Hispanic/African American patients, and CSF2 and CCL5 negatively correlated with the decreased expression of TET3. Interestingly, the expression of gene encoding CD34, a marker of myeloid and lymphoid precursor cells, and CD163, a monocyte/macrophage protein, was higher in the leukocytes of Caucasian than Hispanic/African American patients. Furthermore, Hispanic/African American patients having higher TET2/TET3 expression had higher pulmonary capillary wedge pressure. In conclusion, our results revealed higher DNMT1 and lower TET2/TET3 in Caucasian than Hispanic/African American patients together potentially augmented genes encoding inflammation causing cytokines, and CD34+‐derived immunogenic cells, and the severity of PAH.

Regulation of endothelial DNA methylation by IL‐6 signaling

Interleukin 6 (IL‐6) is a major mediator of the septic cytokine storm. It activates the transcription factor STAT3, resulting in pathological vascular leakage, leukocyte adhesion, and coagulation cascade activation, leading to acute multiorgan dysfunction. Septic shock survivors have a high risk of developing post‐intensive care syndrome (PICS), a condition of chronic cardiovascular disease, cognitive impairment, and reduced overall survival. The causes for this syndrome are not fully understood. We hypothesize that acute STAT3‐mediated endotheliopathy promotes changes in the endothelial DNA methylation landscape, leading to the long‐lasting effects of PICS. To test this in vitro, HUVEC were treated with a combination of IL‐6 and sIL‐6Rα (IL‐6+R) or saline for 6h, 24h, or 72h, and DNA methylation was profiled using Infinium Methylation EPIC arrays after bisulfite conversion. Unbiased clustering showed a separation between treated and control cells only after 72h. 360 CpG sites were significantly modified (FDR‐adjusted p=&lt;0.05). Of these, 329 CpG sites were hypomethylated and 31 were hypermethylated. Gene ontology analysis of the differentially methylated genes showed enrichment of pathways directly linked with the platelet and leukocyte interaction, cell proliferation and angiogenesis, and signaling. Consistent with the demethylation of the LAMP3 locus, a 72h treatment with IL‐6+R induced a dramatic increase in LAMP3 expression. The same treatment induced an inhibition of ADAM19 and MYO10 gene expression, consistent with their hypermethylation status. These changes were not associated with differences in the expression of DNA methyltransferases and TET enzymes. The IL‐6+R‐induced increase in LAMP3 expression was further increased in cells that had been previously treated with IL‐6+R for 72h and then incubated in the absence of IL‐6+R for another 96h, compared to saline‐treated cells, suggesting a long‐lasting effect of a prior IL‐6+R treatment via demethylation of the promoter. In contrast, SOCS3 expression (a gene directly activated by STAT3) was similar in cells that were pretreated with IL‐6+R or not. In summary, persistent activation of IL‐6 leads to endothelial DNA methylation changes and corresponding gene expression differences. These differences may regulate pathways that can be associated with reduced long‐term survival and prolonged organ damage as observed in PICS.

Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells

Abstract Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α. Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125

The Impact of Sorghum Polyphenols on DNA Methylation and Signaling Pathways in Colon Cancer

Colorectal cancer (CRC) is a major public health issue, accounting for nearly 150,000 cases annually. Because CRC correlates with inflammation and oxidative stress, exploiting the anti‐oxidative and anti‐inflammatory nature of bioactive components in our diet, such as polyphenols, could potentially contribute to the inhibition of cancer initiation. Polyphenols are an immense family of naturally occurring secondary plant metabolites found in fruits, vegetables, and cereal grains, and have been demonstrated across many in vitro and in vivo studies to protect against carcinogenesis. Not only are polyphenols implicated to impact cancer development but they may also be involved in epigenetic regulation. Abnormalities in epigenetic modifications can lead to aberrant regulation of gene expression, and depending on the genes in question, this may contribute to the cell’s ability to acquire hallmarks of cancer. New varietals of the grain Sorghum bicolor provided by the USDA contain high amounts of polyphenols, which may contribute to CRC prevention due to their strong antioxidative and anti‐carcinogenic properties. The expression of genes involved in the promotion or prevention of CRC, like the Wnt/β‐catenin signaling pathway, may be regulated via epigenetic mechanisms. We hypothesize that the high polyphenol sorghum extracts may decrease DNA methylation, thus impacting the expression genes involved in the Wnt/β‐catenin signaling pathway. For our current study, we treated HCT116 and SW480 human colon cancer cells with novel high‐polyphenol S. bicolor extracts (HP and SC) or an extract of low‐polyphenol Macia‐Sorghum for up to 48 hours. Controls included cells treated with solvent or 5 µM epigallocatechin gallate as a benchmark. Total RNA was harvested, and the mRNA expression of selected genes was quantitated via qPCR. Relative to solvent control, the high‐polyphenol S. bicolorextract HP increased mRNA expression of DNA methyltransferase 1 (DNMT1), the key enzyme that upholds methylation patterns after DNA synthesis. Furthermore, mRNA expression of DNMT3A, the enzyme responsible for de novo DNA methylation, was also increased. The SC extract significantly decreased the expression of β‐catenin while increasing expression of transcription factor 4 (TCF4), a gene that mediates the expression of pro‐tumorigenic Wnt target genes such as VEGF and cMYC. Encouraged by these preliminary findings, we will investigate mRNA and protein expression of genes that are involved in epigenetic mechanisms, as well as those of downstream signaling pathways. Our study may help elucidate the question, whether consumption of high‐polyphenol S. bicolor as part of the human diet may lead to beneficial changes in DNA methylation. Subsequently, the expression of specific genes involved in the promotion or prevention of colorectal cancer may be modified, providing a dietary intervention to CRC.

SNHG3 Affects Gastric Cancer Development by Regulating SEPT9 Methylation.

Background Gastric cancer (GC) is a common malignancy that can be formed by methylation-induced deactivation of tumor silencer genes, which is one of the key mechanisms of tumorigenesis. SEPT9 methylation, a symptomatic marker for tumors, can downregulate gene expression. Long noncoding RNA small nucleolar host gene 3 (lncRNA SNHG3) is a new type of lncRNA related to cancer. Our study investigated the mechanism of SNHG3 regulation of SEPT9 methylation and its effects on the growth, metastasis, and spread of gastric cancer cells. Methods Quantitative real-time PCR (qRT–PCR) was used to detect SNHG3 and miR-448 in gastric cancer, and a dual-luciferase experiment verified the effects of SNHG3, miR-448, and DNMT1. After abnormally expressing SNHG3, miR-448, and DNMT1 alone or together, methylation-specific PCR was performed to determine the methylation of SEPT9, Western blotting was performed to detect the expression of DNA methyltransferase 1 (DNMT1) and SEPT9, and Transwell, scratch, and CCK-8 assays were performed to reveal the invasion, migration, and cell growth of gastric cancer cells. Results We found that SNHG3 was upregulated in gastric cancer and that SNHG3 knockdown or miR-448 overexpression inhibited SEP9 methylation and therefore increased its expression, thereby inhibiting the growth, metastasis, and spread of gastric cancer cells. Conclusion Our study indicates that SNHG3 regulates SEPT9 methylation by targeting miR-448/DNMT1 and subsequently affecting the occurrence and development of gastric cancer.

Investigating the role of DNMT1 gene expression on myocardial ischemia reperfusion injury in rat and associated changes in mitochondria

Altered DNA methylation and mitochondrial dysfunction are the two key features of myocardial ischemia reperfusion injury (I/R), but their association with I/R remains unknown. In the present study, the relationship between DNA methyl transferase1 (DNMT1), the key methylation gene, and the mitochondrial quality control genes in rat heart during I/R was explored. We used the Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion and subsequent inhibition of DNMT1 with 5-azacytidine to evaluate the role of DNA methylation in I/R. Reperfusion significantly increased the expression of the DNMT1 gene, enzyme activity, and global DNA methylation levels, along with decreased mitochondrial copy, electron transport chain (ETC) activities, and ATP level. This was in agreement with the significant downregulation of 11 mitochondrial genes PGC-1α, TFAM, POLG, MFN1 and MFN2, FIS1, PARKIN, OPTN, ND1, ND4L, Cyt B and COX1 in I/R induced rat hearts. The expression pattern of the mitochondrial genes PGC-1α, TFAM, ND1 and Cyt B showed a significant negative correlation with DNMT1 expression. Rate pressure product, index of cardiac performance negatively correlated with DNMT1 expression (r = -0.8231, p = 0.0456). However, DNMT1 inhibited rat hearts via 5-azacytidine significantly improved the heart from I/R injury and reversed the I/R associated changes in the gene expression of TFAM, POLG, PGC-1α, ND1, COX1 and Cyt B, and improved the overall mtDNA copies, with a subsequent improvement in the ETC enzyme activity and ATP levels. To conclude, I/R augmented the DNMT1 activity with a subsequent increase in cardiac injury via downregulating the mitochondrial functional genes.

The Potential Role of Nigella sativa Seed Oil as Epigenetic Therapy of Cancer.

Nigella sativa oil, commonly known as black seed oil (BSO), is a well-known Mediterranean food, and its consumption is associated with beneficial effects on human health. A large number of BSO’s therapeutic properties is attributed to its pharmacologically active compound, thymoquinone (TQ), which inhibits cell proliferation and induces apoptosis by targeting several epigenetic players, including the ubiquitin-like, containing plant homeodomain (PHD) and an interesting new gene, RING finger domains 1 (UHRF1), and its partners, DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1). This study was designed to compare the effects of locally sourced BSO with those of pure TQ on the expression of the epigenetic complex UHRF1/DNMT1/HDAC1 and the related events in several cancer cells. The gas chromatographs obtained from GC-MS analyses of extracted BSO showed that TQ was the major volatile compound. BSO significantly inhibited the proliferation of MCF-7, HeLa and Jurkat cells in a dose-dependent manner, and it induced apoptosis in these cell lines. BSO-induced inhibitory effects were associated with a significant decrease in mRNA expression of UHRF1, DNMT1 and HDAC1. Molecular docking and MD simulation showed that TQ had good binding affinity to UHRF1 and HDAC1. Of note, TQ formed a stable metal coordinate bond with zinc tom, found in the active site of the HDAC1 protein. These findings suggest that the use of TQ-rich BSO represents a promising strategy for epigenetic therapy for both solid and blood tumors through direct targeting of the trimeric epigenetic complex UHRF1/DNMT1/ HDAC1.

MiR-29b-3p Inhibitor Alleviates Hypomethylation-Related Aberrations Through a Feedback Loop Between miR-29b-3p and DNA Methylation in Cardiomyocytes.

As a member of the miR-29 family, miR-29b regulates global DNA methylation through target DNA methyltransferases (DNMTs) and acts as both a target and a key effector in DNA methylation. In this study, we found that miR-29b-3p expression was inversely correlated with DNMT expression in the heart tissues of patients with congenital heart disease (CHD), but whether it interacts with DNMTs in cardiomyocytes remains unknown. Further results revealed a feedback loop between miR-29b-3p and DNMTs in cardiomyocytes. Moreover, miR-29b-3p inhibitor relieved the deformity of hypomethylated zebrafish and restored the DNA methylation patterns in cardiomyocytes, resulting in increased proliferation and renormalization of gene expression. These results suggest mutual regulation between miR-29b-3p and DNMTs in cardiomyocytes and support the epigenetic normalization of miRNA-based therapy in cardiomyocytes.

MicroRNA-342-3p regulates yak oocyte meiotic maturation by targeting DNA methyltransferase 1.

MicroRNAs (miRNAs) play vital roles in the development of oocytes and ovarian follicles. We have previously shown differential expression of miR-342-3p during yak oocyte maturation. In this study, we investigated the role of miR-342-3p in meiotic maturation of yak oocytes and the underlying mechanism. The profile of ovarian DNA methyltransferase 1 (DNMT1) expression was investigated in yak by RT-qPCR and western blot analyses. The pattern of Dnmt1 expression in various meiotic stages (GV stage, MI stage and MII stage) of yak oocyte maturation was then measured by immunofluorescence staining. The interaction between Dnmt1 and miR-342-3p was verified by dual-luciferase reporter assay. Finally, miR-342-3p inhibitors were microinjected into yak cumulus-oocyte complex to evaluate the effects on oocyte maturation. MiR-342-3p expression was upregulated in oocytes during meiotic maturation, with significantly higher levels in the MII stage compared with the GV- and MI stages (p<.05), whereas the opposite pattern of Dnmt1 expression was detected. In the period to sexual maturity (3-year-old), DNMT1 showed an age-related pattern of ovarian expression at both the gene and protein levels. Immunohistochemistry analysis also indicated maturation-stage-related differences in DNMT1 expression in the ovarian follicles and corpus luteum, with expression predominantly detected in cumulus cells and oocytes. MiR-342-3p inhibitors effectively upregulated Dnmt1 expression and significantly inhibited oocyte meiotic maturation. Taken together, our results indicate that miR-342-3p plays a vital role in the meiotic maturation of yak oocytes by targeting the 3'-untranslated regions (UTR) of Dnmt1 and provide a new perspective on the mechanism of this process.