DNA Methyltransferase 3A Levels Research Articles

Decreased SUV39H1 at the promoter region leads to increased CREMα and accelerates autoimmune response in CD4+ T cells from patients with systemic lupus erythematosus

BackgroundOverproduction of cAMP-responsive element modulator α (CREMα) in total T cells from patients with systemic lupus erythematosus (SLE) can inhibit IL-2 and increase IL-17A. These ultimately promote progression of SLE. This study aims to investigate the expression of CREMα in SLE CD4+ T cells and find out the mechanisms for the regulation of CREMα in SLE CD4+ T cells.ResultsCREMα mRNA was overexpressed in CD4+ T cells from SLE patients. The levels of histone H3 lysine 9 trimethylation (H3K9me3) and suppressor of variation 3–9 homolog 1 (SUV39H1) at the CREMα promoter of SLE CD4+ T cells were markedly decreased. Down-regulating SUV39H1 in normal CD4+ T cells elevated the levels of CREMα, IL-17A, and histone H3 lysine 4 trimethylation (H3K4me3) in the CREMα promoter region, and lowered IL-2, H3K9me3, DNA methylation, and DNA methyltransferase 3a (DNMT3a) enrichments within the CREMα promoter, while no sharp change in SET domain containing 1 (Set1) at the CREMα promoter. Up-regulating SUV39H1 in SLE CD4+ T cells had the opposite effects. The DNA methylation and DNMT3a levels were obviously reduced, and H3K4me3 enrichment was greatly increased at the CREMα promoter of CD4+ T cells from SLE patients. The Set1 binding in the CREMα promoter region upgraded significantly, and knocking down Set1 in SLE CD4+ T cells alleviated the H3K4me3 enrichment within this region, suppressed CREMα and IL-17A productions, and promoted the levels of IL-2, CREMα promoter DNA methylation, and DNMT3a. But there were no obviously alterations in H3K9me3 and SUV39H1 amounts in the region after transfection.ConclusionsDecreased SUV39H1 in the CREMα promoter region of CD4+ T cells from SLE patients contributes to under-expression of H3K9me3 at this region. In the meantime, the Set1 binding at the CREMα promoter of SLE CD4+ T cells is up-regulated. As a result, DNMT3a and DNA methylation levels alleviate, and H3K4me3 binding increases. All these lead to overproduction of CREMα. Thus, the secretion of IL-2 down-regulates and the concentration of IL-17A up-regulates, ultimately promoting SLE.

Impact of the interaction between the polymorphisms and hypermethylation of the CD36 gene on a new biomarker of type 2 diabetes mellitus: circulating soluble CD36 (sCD36) in Senegalese females

BackgroundSeveral predisposing factors for diabetes mellitus have been identified, including cluster determinant 36 (CD36) receptor expression. We aimed to determine the effects of CD36 gene polymorphisms and hypermethylation on the plasma CD36 protein levels in type 2 diabetes.Materials and methodsWe conducted a cross-sectional study involving 100 females (lean healthy control subjects and subjects with type 2 diabetes). This study was conducted at the Human Physiology Laboratory at the Dakar Faculty of Medicine in Senegal. Circulating sCD36 levels and DNA methyltransferase 3a levels were determined by enzyme-linked immunosorbent assay. The other biological parameters were evaluated in a biochemical laboratory. CD36 gene polymorphisms and methylation were explored by real-time polymerase chain reaction and methylation-specific polymerase chain reaction, respectively.ResultssCD36 was negatively correlated with HDL-cholesterol levels (r = − 0.52 p = 0.0001) and triglyceride levels (r = − 0.36 p = 0.01) in control subjects. However, in the type 2 diabetes group, sCD36 levels were positively correlated with total cholesterol levels (r = 0.28 p = 0.04). For rs3211867, control subjects harboring the CC genotypes had significantly higher sCD36 levels than control subjects harboring the AA/AC genotype (p = 0.02); in the type 2 diabetes group, the sCD36 level was not significantly lower in subjects harboring the AA/AC genotype than in subjects harboring the CC genotype (p = 0.27). CD36 gene methylation reduced the sCD36 level in the control subjects compared to control subjects without CD36 gene methylation (p = 0.03). This difference was not significant in the type 2 diabetes group comparing subjects with diabetes with CD36 gene methylation to subjects with diabetes without CD36 gene methylation (p = 0.09). We noted a nonsignificant increase in sCD36 levels in subjects with diabetes with CD36 gene methylation compared to control subjects with CD36 gene methylation (p = 0.27). A combination of the CD36 polymorphism effect and the CD36 methylation effect did not significantly reduce sCD36 levels in subjects with type 2 diabetes.ConclusionCD36 gene polymorphisms and CD36 gene methylation separately reduce sCD36 levels. Their impacts are compensated for in subjects with type 2 diabetes by an increase in sCD36 levels, the mechanism of which needs to be elucidated.

Association between polymorphisms and hypermethylation of CD36 gene in obese and obese diabetic Senegalese females

BackgroundObesity and related metabolic disorders are associated with genetic and epigenetic alterations. In this study, we have examined the association between polymorphisms and hypermethylation of the CD36 gene promoter with obesity in Senegalese females with or without type 2 diabetes mellitus to identify novel molecular markers of these pathologies (obesity and type 2 diabetes mellitus).Materials and methodsThe study was conducted in Senegal with healthy lean control, obese, and obese diabetic (age; 49.98 years ± 7.52 vs 50.50 years ± 8.76 vs 51.06 ± 5.78, and body mass index (BMI); 24.19 kg/m2 ± 2.74 vs 34.30 kg/m2 ± 4.41 vs 33.09 kg/m2 ± 4.30). We determined three genetic polymorphisms of CD36 i.e., rs1761667, rs1527483, and rs3211867 by real-time polymerase chain reaction, and methylation of CPG islands of CD36 was assessed by methylation-specific polymerase chain reaction (MS-PCR) in DNA isolated from peripheral blood of each participant. Plasma sCD36 levels and DNA methyltransferase 3a (DNMT3a) levels were determined by enzyme-linked immunosorbent assay (ELISA). According to the standard laboratory protocol, all biochemical parameters were analyzed from fasting serum or plasma.ResultsFor rs1761667, obese and obese diabetic subjects had statistically significant different parameters depending on the genotypic distribution. These were waist size for obese and HDL cholesterol for obese diabetic, they were significantly higher in subjects harboring GG genotype of rs1761667 (respectively p = 0.04 and p = 0.04).For rs3211867, obese subjects harboring the AA/AC genotype had significantly higher BMI (p = 0.02) and total cholesterol (p = 0.03) than obese subjects harboring the CC genotype. At the same time, the obese diabetic subjects harboring the AA/AC genotype had total cholesterol levels significantly higher than the obese diabetic subjects harboring the CC genotype (p = 0.03). For rs1527483, only the control subjects had statistically significant different parameters depending on the genotypic distribution. The control subjects harboring the GG genotype had a significantly higher BMI than the control subjects harboring the AA/AG genotype (p = 0.003). The CD36 gene methylation was significantly 1.36 times more frequent in obese and obese diabetic compared to lean control (RR = 1.36; p = 0.04). DNMT3a levels were higher in subjects with CD36 gene methylation than in subjects without CD36 gene methylation in each group. Obese diabetic subjects with CD36 gene methylation had significantly fewer plasmas sCD36 (p = 0.03) and more LDL-cholesterol (p = 0.01) than obese diabetic subjects without CD36 gene methylation. In the control group, an increase in sCD36 levels would be associated with a decrease in total cholesterol and triglyceride levels (coef = -7647.56 p = 0.01 and coef = -2528.50 p = 0.048, respectively) would be associated with an increase in LDL cholesterol levels. For the obese group, an increase in sCD36 levels would be associated with an increase in fasting insulin levels (coef = 490.99 p = 0.02) and a decrease in glycated hemoglobin levels (coef = -1196.26 p = 0.03). An increase in the sCD36 levels would be associated with an increase in the triglyceride levels in the obese diabetic group (coef = 9937.41 p = 0.02). The AA/AC genotype of SNP rs3211867 polymorphism was significantly associated with CD36 gene methylation in the control and obese diabetic groups (respectively p = 0.05, p = 0.002; 95% CI).ConclusionThese observations suggest that polymorphisms and epigenetic changes in CD36 gene promoters may be implicated in the onset of obesity and its related complication type 2 diabetes mellitus.

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity

To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Downregulation of DNMT3a expression by RNAi and its effect on NF-κBs expression of thymic epithelial cells

ObjectiveTo understand the characteristics of DNA methyltransferase 3a (DNMT3a) in thymoma associated Myasthenia Gravis reveal its transcriptional regulator network as while as analyze the effect of DNMT3a on Rel/ nuclear factor-kappaB family (RelA/RelB) and its downstream autoimmune regulatory factor (Aire). MethodsTissues of 30 patients with thymoma, with or without myasthenia gravis (MG), were collected and the DNMT3a protein expression were evaluated through immunohistochemistry. We performed mRNA expression profiling microarray detection and analysis, and integrated the analysis by constructing protein-protein interaction networks and the integration with other database. We identified molecular difference between low and high DNMT3a in the thymoma by heatmap. We also performed PCR validation in thymoma tissues. The DNMT3a-shRNA plasmid was transfected into TEC cells, and these cells were treated with 5-aza-2-deoxycytidine, a blocker of DNMT3a. After the down-regulation of DNMT3a in TEC cells, the transcript and protein levels of RelA, RelB, Aire, and CHRNA3 were evaluated by western blotting. In addition, changes in gene expression profiles were screened through microarray technology. We performed differential gene analysis in the thymoma cohort by heatmap with R (v.4.3.0) software. ResultsIn 30 matched tissue specimens, the expression of DNMT3a protein in thymoma with MG was lower than that in thymoma. Through mRNA expression profiling analysis, we constructed a co-expression network of DNMT3a and found direct interaction between IKZF1 and DNMT3a, and this co-expression relationship was overlappted with Cistrome DB database. We found up-regulation of 149 mRNAs and repression of 177 mRNAs in thymoma with MG compared with thymoma. Gene ontology and pathway analysis show the involvement of a multitude of genes in the mis-regulation of MG-related pathways. RNA interference significantly reduced the level of mRNA of DNMT3a, which proved that plasmid DNMT3a was effective. In comparison to the control group, the levels of DNMT3a, Aire, and CHRNA3 mRNA and protein in TEC cells transfected with DNMT3a-shRNA interference plasmid were significantly decreased, while the expression level of RelA and RelA/RelB was significantly increased. ConclusionsOur study reveals the DNMT3a-NF-κB pathway has a major effect on MG, and can be used as a marker for diagnosis as well as a target for MG treatment.

Cigarette smoke extract promotes DNA methyltransferase 3a expression in dendritic cells, inducing Th-17/Treg imbalance via the c-Jun/allograft inflammatory factor 1 axis.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disorder. Although numerous studies on COPD have been conducted, therapeutic strategies for COPD are limited, and its pathological mechanism is still unclear. The present study aimed to explore the role of DNA methyltransferase 3a (DNMT3a) in dendritic cells (DCs) and the possible role of the Th-17/Treg cell balance in COPD. Immature DCs (iDCs) were induced and cocultured with CD4+ T cells. An in vitro COPD model was established by treatment with cigarette smoke extract (CSE). DNMT3a or allograft inflammatory factor 1 (AIF1) and c-Jun N-terminal kinase (JNK) were inhibited and overexpressed, respectively, by transfection with sh-DNMT3a or sh-AIF1 and JNK overexpression plasmids. The 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell viability. The Th17/Treg cell ratio was determined by flow cytometry. The expression levels of DNMT3a, c-Jun and AIF1 were measured using RT-qPCR or western blotting. Chromatin immunoprecipitation (CHIP) was used to confirm the interaction between c-Jun and the AIF1 promoter region. CSE stimulation promoted the expression of DNMT3a, and AIF1, and the ratio of p-c-Jun/c-Jun in iDCs. Besides, the iDC-mediated differentiation of Th17 cells was in a dose-dependent manner. However, knockdown of DNMT3a or AIF1 reversed the above effects caused by CSE. Inhibition of c-Jun signaling by treatment with the JNK inhibitor SP600125 also suppressed the iDC-mediated differentiation of Th17 cells, which was promoted by CSE. CHIP analysis showed that c-Jun could bind to the promoter region of AIF1. DNMT3a could regulate the iDC-mediated Th17/Treg balance by regulating the c-Jun/AIF1 axis.

Environmental enrichment enhances sociability by regulating glutamate signaling pathway through GR by epigenetic mechanisms in amygdala of Indian field mice Mus booduga

Environmental enrichment (EE) dynamically regulates gene expression and synaptic plasticity with positive consequences on behavior. The present study was performed on field-mice to explore the effects of EE on both captive-condition inducing social stress and epigenetic changes of molecules resilience stress. For this purpose, field-mice were caught and allowed to habituate in standard laboratory conditions for 7 days. The next day animals were randomly assigned to three groups: i) mice at short-term standard condition (STSC); which were subjected to social interaction test (SIT) on day 9, ii) mice continuously maintainedfor additional 30 days, with these long-term standard conditions (LTSC), and iii) mice maintained in an EE cage for additional 30 days. After achieving SIT, we examined epigenetic changes of a repertory of molecules associated with resilience stress, by determining their levels by Western blot. Thus, the main findings were that during SIT, EE exerted more social interaction of field-mice with the strangers compared with STSC and LTSC mice. Related with social behavior results, we found that in mice subjected to EE the levels of histone 3 lysine 9 di-methylation (H3K9me2), glucocorticoid receptor (GR), N-methyl-D asparate (NMDA) receptor subunits NR2A and NR2B, postsynaptic density protein-95 (PSD-95), and mature brain-derived neurotrophic factor (mBDNF) were significantly elevated; whereas the levels of DNA methyltransferase-3A (DNMT3A), methyl-CpG-binding protein-2 (MeCP2), repressor element-1 silencing transcription factor (REST), H3K4me2 and lysine demethylase-1A (KDM1A) decreased. These results suggest that enhanced sociability of EE mice could be mediated, in part, by altered expression of molecules regulating glutamate signaling pathway through GR by epigenetic mechanisms.

Cuscuta chinensis flavonoids down-regulate the DNA methylation of the H19/Igf2 imprinted control region and estrogen receptor alpha promoter of the testis in bisphenol A exposed mouse offspring.

Exposure to the emerging contaminant bisphenol A (BPA) is ubiquitous and associated with reproductive disorders. The BPA effect as an endocrine disruptor is widely known but other mechanisms underlying developmental disease, such as epigenetic modifications, still remain unclear. The objective of this study was to investigate whether Cuscuta chinensis flavonoids (CCFs) can be used as a dietary supplement to reverse BPA-induced epigenetic disorders, by analyzing the molecular processes related to BPA impairment of testicular development. BPA and different concentrations of CCFs were administered to the dams at gestation day (GD) 0.5-17.5. The testis and serum of male mice were collected at postnatal day (PND) 21 and PND 56 for the detection of related indicators. Our results showed that compared with the BPA group, CCFs could significantly increase the serum contents of testosterone (T), estradiol (E2) in males at PND 21 and PND 56, as well as the contents and transcription levels of DNA methyltransferase 3A (Dnmt3A), Dnmt3B in males at PND 21 and that of estrogen receptor alpha (ERα) at PND 56. The expressions of Dnmt1 and ERα at PND 21 and ERβ at both PND 21 and PND 56 in males were significantly decreased with the administration of different concentrations of CCFs (P < 0.01 or P < 0.05). CCFs also significantly inhibited the BPA-induced hypermethylated status of the ERα promoter and H19/Igf2 imprinting control region (ICR) in the testis at PND 56. These results indicated that CCFs could decrease the methylation levels of ERα and H19/Igf2 genes by inhibiting the expression of DNA methyltransferases (DNMTs), thereby decreasing the levels of reproductive hormones and receptors in adult males, and ultimately alleviating the negative effect of BPA on testicular development in male mice.

Active Amyloid-β Vaccination Results in Epigenetic Changes in the Hippocampus of an Alzheimer's Disease-Like Mouse Model.

While evidence accumulates for a role of epigenetic modifications in the pathophysiological cascade of Alzheimer's disease (AD), amyloid-β (Aβ)-targeted active immunotherapy approaches are under investigation to prevent or slow the progression of AD. The impact of Aβ active vaccines on epigenetic markers has not been studied thus far. The current study aims to establish the relationship between active immunotherapy with a MER5101-based vaccine (consisting of Aβ1-15 copies conjugated with a 7 aa spacer to the diphtheria toxoid carrier protein, formulated in a Th2-biased adjuvant) and epigenetic DNA modifications in the hippocampus of APPswe/PS1dE9 mice. As we previously reported, immunotherapy started when the mice were 10 months of age and behavioral testing occurred at 14 months of age, after which the mice were sacrificed for further analysis of their brains. In this add-on study, global levels of DNA methylation and hydroxymethylation, and DNA methyltransferase 3A (DNMT3A) were determined using quantitative immunohistochemistry, and compared to our previously analyzed immunization-induced changes in AD-related neuropathology and cognition. Active immunization did not affect global DNA methylation levels but instead, resulted in decreased DNA hydroxymethylation and DNMT3A levels. Independent of immunization, inverse correlations with behavioral performance were observed for levels of DNA methylation and hydroxymethylation, as well as DNMT3A, while Aβ pathology and synaptic markers did not correlate with DNA methylation levels but did positively correlate with DNA hydroxymethylation and levels of DNMT3A. Our results indicate that active Aβ vaccination has significant effects on the epigenome in the hippocampus of APPswe/PS1dE9 mice, and suggest that DNA methylation and hydroxymethylation may be involved in cognitive functioning.

5-Aza-2'-deoxycytidine in the medial prefrontal cortex regulates alcohol-related behavior and Ntf3-TrkC expression in rats.

Recent studies have indicated that DNA methylation plays an important role in the development of alcohol abuse. 5-Aza-2’-deoxycytidine (5-Aza-dc), an inhibitor of DNA methyltransferases, was FDA approved for myelodysplastic syndrome treatment. However, it is unclear whether 5-Aza-dc is involved in alcohol abuse. In this study, using a chronic alcohol exposure model in rats, 5-Aza-dc was injected into the medial prefrontal cortex (mPFC). Alcohol-drinking behavior and the anxiety related behavior were evaluated by two-bottle choice and open field test. We found that 5-Aza-dc injection into the mPFC significantly decreased alcohol consumption and alcohol preference in alcohol-exposure rats, corresponding to the reduced blood alcohol levels. Although 5-Aza-dc potentiated the anxiety-like behavior of alcohol-exposure rats, it had no effect on the locomotor activity. Moreover, both of the mRNA and protein levels of DNA Methyltransferase 3A (DNMT3A) and DNMT3B in the mPFC were upregulated after 35 days of alcohol exposure and this upregulation could be reversed by 5-Aza-dc treatment. Additionally, 5-Aza-dc reversed the alcohol-induced downregulation of neurotrophin-3 (Ntf3), correspondingly the expression of its receptor-TrkC was reduced. These findings identified a functional role of 5-Aza-dc in alcohol-related behavioral phenotypes and one of the potential target genes, Ntf3. We also provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcohol abuse.

Metastasis-associated protein 1 is an upstream regulator of DNMT3a and stimulator of insulin-growth factor binding protein-3 in breast cancer

Despite a recognized role of DNA methyltransferase 3a (DNMT3a) in human cancer, the nature of its upstream regulator(s) and relationship with the master chromatin remodeling factor MTA1, continues to be poorly understood. Here, we found an inverse relationship between the levels of MTA1 and DNMT3a in human cancer and that high levels of MTA1 in combination of low DNMT3a status correlates well with poor survival of breast cancer patients. We discovered that MTA1 represses DNMT3a expression via HDAC1/YY1 transcription factor complex. Because IGFBP3 is an established target of DNMT3a, we investigated the effect of MTA1 upon IGFBP3 expression, and found a coactivator role of MTA1/c-Jun/Pol II coactivator complex upon the IGFBP3 transcription. In addition, MTA1 overexpression correlates well with low levels of DNMT3a which, in turn also correlates with a high IGFBP3 status in breast cancer patients and predicts a poor clinical outcome for breast cancer patients. These findings suggest that MTA1 could regulate the expression of IGFBP3 in both DNMT3a-dependent and -independent manner. Together findings presented here recognize an inherent role of MTA1 as a modifier of DNMT3a and IGFBP3 expression, and consequently, the role of MTA1-DNMT3a-IGFBP3 axis in breast cancer progression.

Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells.

To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC50) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G0/G1 phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

EID3 directly associates with DNMT3A during transdifferentiation of human umbilical cord mesenchymal stem cells to NPC-like cells

There has been recently been increased interest in the plasticity of human umbilical cord mesenchymal stem cells (UMSCs) and their potential in the treatment of neurological disorders. In this study, UMSCs were transdifferentiated into neural stem-like cells (uNSCL), these cells grow in neurosphere-like structures and express high levels of NSCs markers. Epigenetics-related gene screening was here used to assess the relationship between E1A-like inhibitor of differentiation 3 (EID3), a p300 inhibitor, and DNA methyltransferase 3 A (DNMT3A) during the transdifferentiation of UMSCs into uNSCL in vitro. Before transdifferentiation of UMSCs into uNSCLs, high levels of EID3 and low levels of DNMT3A were detected; after transdifferentiation, low levels of EID3 and high levels of DNMT3A were detected. The current work showed that EID3 and DNMT3A co-localized in cell nuclei and EID3 interacted directly with DNMT3A in uNSCL. In summary, these results suggest that DNMT3A is probably directly regulated by EID3 during UMSC transdifferentiation into uNSCLs. These findings indicated a novel mechanism by which EID3, a p300 acetyltransferase inhibitor, could directly affect DNMT3A, this enzyme possesses dual methylation and demethylation abilities. These studies may be helpful for understanding a complex regulation mode of DNMT3A, which is a unique member of the methyltransferase family.

MicroRNA-200b inhibits the proliferation of hepatocellular carcinoma by targeting DNA methyltransferase 3a.

Aberrant microRNA (miRNA or miR) expression has been reported to contribute to the pathogenesis of hepatocellular carcinoma (HCC). However, the involvement of specific miRNAs in HCC remains to be elucidated. The present study aimed to investigate the potential role of miR-200b and the mechanism underlying its function in hepatocarcinogenesis. The results of the present study demonstrated that the expression levels of miR‑200b were significantly reduced in HCC tissue samples, as compared with normal liver (NL) and para‑tumorous (PT) tissue samples. The results also revealed that miR‑200b expression levels in HepG2 cells were significantly decreased compared with those in L02 cells. In addition, western blotting and reverse transcription‑quantitative polymerase chain reaction demonstrated that the expression levels of DNA methyltransferase3a (DNMT3a), a possible target gene for miR‑200b, were significantly higher in HCC tissue samples, as compared with those in NL and PT tissue samples. Furthermore, the data suggested that DNMT3a was a direct target gene of miR‑200b. Upregulated miR‑200b expression in HepG2 cells led to a decrease in DNMT3a expression levels, and an inhibition of cell proliferation. These results suggested that miR‑200b has an important role in hepatocarcinogenesis and acts by downregulating DNMT3a expression. Thus, miR-200b may be a promising target for HCC treatment.

Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

BackgroundEpithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.AimsTo evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.Materials and MethodsDNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin), DNA methyltransferase 3a (DNMT3a) and NFκB (for treated HDFα cells).ResultsAdministration of tumor derived DNA on HT29 cells resulted in significant (p<0.05) mRNA level alteration in 118 genes (logFc≥1, p≤0.05), including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A), metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1), tumor biomarker (CEACAM5), metabolic genes (i.e. INSIG1, LIPG), messenger molecule genes (i.e. DAPP, CREB3L2). Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05), including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β), STING pathway (ADAR, IRF7, CXCL10, CASP1) and the FGF2 gene.ConclusionsDNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling pathway in normal fibroblasts.

MiR-29b is an indicator of prognosis in breast cancer patients.

MicroRNA-29b (miR-29b) targets numerous important genes that mediate carcinogenesis and tumor development in breast cancer in vitro and in vivo. The aim of the present study was to determine the clinical significance of miR-29b expression in primary breast cancer patients. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of miR-29b and certain target genes of miR-29b, such as DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 1 (TET1) and thymine DNA glycosylase (TDG), was performed in 94 primary breast cancer samples. Low expression of miR-29b in primary tumors was significantly associated with poorer disease-free survival (DFS) (P=0.0075) and overall survival (OS) (p=0.0012). Multivariate analysis indicated that miR-29b expression was an independent prognostic factor for OS [relative risk=15.6 (2.33-348), P=0.0026]. In addition, a significant inverse correlation was identified between the expression levels of DNMT3A and miR-29b in estrogen receptor-positive breast cancer patients (P=0.027). To the best of our knowledge, this is the first study to investigate the clinicopathological significance of miR-29b in breast cancer cases and miR-29b is shown to act as a tumor suppressive microRNA in breast cancer and as a potential marker for recurrence and metastasis in breast cancer patients.

Cigarette smoke condensate and individual constituents modulate DNA methyltransferase expression in human liver cells.

Objectives:Previous studies found higher expression levels of DNA methyltransferase 1 in liver samples from smokers compared to those from non-smokers. In contrast, expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were similar in smokers and non-smokers. This study extends these studies to establish a causal linkage to cigarette smoke exposure by examining whether DNA methyltransferase expression is modulated by cigarette smoke condensate.Methods:These experiments were conducted in an in vitro system using HepG2 human liver cells. The dose range of cigarette smoke condensate was 0.1–120 µg/mL. The duration of exposure was up to 72 h.Results:In a 24-h exposure, DNA methyltransferase 1 expression was found to increase significantly in a dose-dependent manner (greater than threefold at 100 µg/mL cigarette smoke condensate). Expression levels of DNA methyltransferase 3a and DNA methyltransferase 3b were, however, not affected under these conditions. The effect of two cigarette constituents, nicotine and cotinine, on DNA methyltransferase 1 expression was also examined. Nicotine exposure significantly increased DNA methyltransferase 1 expression in a dose-dependent manner (greater than twofold at 50 µM). However, under these conditions, cotinine did not increase DNA methyltransferase 1 expression.Conclusion:These results clearly provide additional support of the modulating effect of cigarette smoke on DNA methyltransferase 1 expression. Given the potential of alterations in DNA methyltransferase expression to affect cellular function, this pathway may play a critical role in cigarette smoke-induced toxicity.

Effect of Dynamic DNA Methylation and Histone Acetylation oncPouVExpression in Differentiation of Chick Embryonic Germ Cells

As a crucial pluripotency-related factor, the epigenetic regulation of Oct4 has been studied intensively in mammalians. However, its dynamic changes of DNA methylation and histone modification in avians remain poorly understood. In the present study, we first described the alterations of DNA methylation and histone acetylation in the promoter of chicken PouV (cPouV; the homologue of Oct4 in avian) during chick embryonic germ (EG) cell differentiation. The epigenetic modification analysis showed that DNA methylation in the cPouV promoter increased obviously, while histone acetylation decreased dramatically detected by chromatin immunoprecipitation assay in the process of differentiation. Gene expression analysis detection indicated that the levels of DNA methyltransferase 3a (Dnmt 3a), Dnmt 3b, and histone deacetylase 3 (HDAC 3) transcripts were significantly high, whereas the relative abundance of Dnmt 1, histone acetyltransferase (HAT), and cPouV mRNA was significantly decreased during the conversion of EG to embryoid body-like structures (EBs), which was correlated with the increased level of methylation and reduced level of H3 acetylation. Moreover, in vitro methylation assay indicated that the reporter gene was remarkably inhibited by the methylated promoter of cPouV. To further understand the effect of epigenetic modifiers on cPouV expression, we performed an analysis of EB cells treated with trichostatin A (TSA), Aza-2'-deoxycytidine (Aza), or TSA plus Aza (TSA/Aza). We observed that the effect of TSA/Aza is more sensitive to the reactivation of cPouV compared with TSA or Aza, indicating that these epigenetic inhibitors can function synergistically to facilitate the reprogramming process. The present study provided evidences that a critical role for cPouV activation/repression by DNA methylation and/or histone modifications is involved in the pluripotency maintenance and differentiation process of chick EG.

Gene expression profiling of the DNMT3A R882 mutation in acute leukemia

DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression. DNMT3A mutations and deletions have been previously observed in acute myeloid leukemia (AML), myelodysplastic sydromes and myeloproliferative neoplasms. However, the involvement of DNMT3A in acute lymphoblastic leukemia (ALL) has rarely been reported. In the present study, PCR and direct sequencing was performed to analyze mutations of DNMT3A amino acid residue 882 in 99 acute leukemia patients, including 57 AML patients, 41 ALL patients and a single biphenotypic acute leukemia (BAL) patient. DNMT3A expression was detected in mono-nuclear cells of the bone marrow in these patients and in normal individuals using real-time quantitative polymerase chain reaction, and 17.5% (10/57) of AML patients were found to exhibit DNMT3A mutations. Four missense mutations were observed in the DNMT3A-mutated AML patients, including R882 mutations and a novel single nucleotide polymorphism resulting in the M880V amino acid substitution. However, the ALL and BAL patients were not found to exhibit DNMT3A mutations. The DNMT3A expression levels in the AML patients were significantly higher compared with those of the ALL patients or normal controls. The reduced expression levels of DNMT3A were associated with a significantly lower complete remission rate in the AML patients. However, in the ALL patients, no statistical significance was identified. The results of the present study indicate that DNMT3A may play varying roles in the regulation of DNA methylation in AML and ALL.