mRNA Expression Of DNA Methyltransferases Research Articles

Adverse maternal environment leads to cardiac fibrosis in adult male mice.

Cardiac fibrosis is a cardinal feature of multiple types of cardiovascular disease, which lead to heart failure. Multiple studies connect adverse maternal environment (AME) with cardiac fibrosis. AME does not always result in fibrosis, though. An additional "insult", such as an adult Western diet (WD), is frequently necessary. The additive effects of AME and adult WD on cardiac fibrosis is not well-understood. AME can also alter DNA methylation. DNA methyltransferase (DNMT) and ten-eleven translocation (TET) are methylation modifying genes that regulate DNA methylation, but it is unknown if AME changes cardiac gene expression of DNMT and TET. We sought to use a model of AME and adult WD to investigate the development of cardiac fibrosis and cardiac mRNA expression of DNMT and TET genes. We exposed dams to WD or control diet (CD) 5 weeks before pregnancy and through lactation. We added environmental stressors during the last third of pregnancy to dams on WD to create AME. Dams on CD experienced no added stressors to create control maternal environment (CME). Male offspring were weaned at Postnatal Week 3 (W3) and placed on WD or CD to create four groups: CME-CD, CME-WD, AME-CD, and AME-WD. AME-WD increased cardiac fibrosis in adulthood (p < .05), whereas AME-CD and CME-WD did not. TET1-3 and DNMT3a mRNA levels decreased in AME versus CME offspring (p < .01). AME increases susceptibility to cardiac fibrosis in adult male mice. Early-life changes to TET expression may mediate susceptibility to fibrosis, but further testing is needed.

Dynamic DNA Methylation Changes of Tbx21 and Rorc during Experimental Autoimmune Uveitis in Mice.

The key transcription factors of T helper cell subpopulations, including T-bet, GATA3, RORγt, and Foxp3 are involved in various autoimmune diseases. Whether methylation of these master transcription factors is associated with the development of experimental autoimmune uveitis (EAU) and the possible epigenetic regulatory mechanisms involved has however not yet been addressed. In our study, significant methylation changes in both Tbx21 and Rorc were observed in one CpG site in the retinas of EAU mice. Two CpG sites of Tbx21 and one CpG site of Rorc showed significant dynamic methylation changes in the RPE-choroid complex during EAU. The mRNA expressions of Tbx21 and Rorc in both the retinas and RPE-choroid complexes correlated with the methylation changes at the various time points during EAU development. The methylation changes were associated with the production of the Th1/Th17 cells' signature cytokines, IFN-γ and IL-17. Dynamic changes in mRNA expression of DNA methyltransferases (DNMT1) were also noted, which may be related to the observed methylation changes of these genes. The present study provides evidence that DNA methylation of Tbx21 and Rorc may be associated with the development of EAU. DNMT1 activation may have an important effect on regulating DNA methylation dynamics.

Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242µM) than in MDA MB 231 (IC50: 0.346-0.37µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

Early-life exposure to three size-fractionated ultrafine and fine atmospheric particulates in Beijing exacerbates asthma development in mature mice

BackgroundEpidemiological studies have suggested that elevated levels of air pollution contribute to an increased incidence or severity of asthma. Although late-onset adult asthma seems to be more attributable to environmental risk factors, limited data is available on the impact of early-life exposure to size-fractionated ambient particulate matter (PM) on asthma in adults. We aimed to determine the effect on the development and exacerbation of asthma in the adult after the mice were exposed as juveniles to three size-fractionated ambient particulates collected from Beijing.MethodsThe three size-fractionated ambient particulates were collected from urban Beijing in winter, heavily affected by traffic and coal-fired emissions. The typical morphological and major chemical components of the PM were characterized first. Oxidative stress and expression of DNA methyltransferases (DNMTs) were then examined in vitro and in the lungs of mouse pups 48 h after exposure to PM by oropharyngeal aspiration. When the exposed and control juvenile mice matured to adulthood, an antigen-induced asthma model was established and relevant bio-indices were assessed.ResultsPM with different granularities can induce oxidative stress; in particular, F1, with the smallest size (< 0.49 μm), decreased the mRNA expression of DNMTs in vitro and in vivo the most significantly. In an asthma model of adult mice, previous exposure as juveniles to size-fractionated PM caused increased peribronchiolar inflammation, increased airway mucus secretion, and increased production of Th2 cytokines and chemokines. In general, F1 and F2 (aerodynamic diameter < 0.95 μm) particulates affected murine adult asthma development more seriously than F3 (0.95–1.5 μm). Moreover, F1 led to airway inflammation in the form of both increased neutrophils and eosinophils in BALF. The activation of the TGF-β1/Smad2 and Smad3/Stat3 signaling pathways leading to airway fibrosis was more profoundly induced by F1.ConclusionThis study demonstrated that exposure to ambient PM in juvenile mice enhanced adult asthma development, as shown by increased Th2 responses, which might be associated with the persistent effects resulting from the oxidative stress and decreased gene expression of DNMTs induced by PM exposure. The observed differences between the effects of three size-fractionated particulates were attributed to particle sizes and chemical constituents, including heavy metals and also PAHs, since the amounts of PAH associated with more severe toxicity were enriched equivalently in the F1 and F2 fractions. Relative to the often mentioned PM2.5, PM with an aerodynamic diameter smaller than 0.95 μm had a more aggravating effect on asthma development.

Effects of folic acid supplementation on cognitive function and Aβ-related biomarkers in mild cognitive impairment: a randomized controlled trial.

Observational studies have frequently reported that low blood folate concentrations are associated with poor cognitive performance. Our previous studies have shown the potential beneficial effect on the metabolite levels of methionine cycle and peripheral blood inflammatory cytokines from 6- and 12-month folic acid supplementation on cognitive function in mild cognitive impairment (MCI). This study aims to continue exploring the effect of 24-month folic acid supplementation on cognitive function and pathological mechanism in MCI. 180 individuals with MCI were identified and randomly divided into intervention (folic acid 400µg/day, n = 90) and convention (n = 90) groups. Cognitive function (WAIS-RC) and blood Aβ-related biomarkers were measured at baseline and at 6, 12, 18, and 24 months. Data were analyzed using generalized estimating equation. This trial has been registered with Trial Number: ChiCTR-TRC-13003227. During the follow-up, scores of full scale IQ, verbal IQ, and subdomains of Information and Digit Span were significantly higher in the intervention group than those in the convention group (P < 0.05). In the intervention group, blood homocysteine, S-adenosylhomocysteine (SAH), Aβ-42, and the expression of APP-mRNA were decreased (P < 0.05), while S-adenosylmethionine (SAM), SAM/SAH ratio, and the expression of DNA methyltransferase mRNA were increased (P < 0.05). Folic acid supplementation appears to improve cognitive function and reduce blood levels of Aβ-related biomarkers in MCI. Larger-scale double-blind placebo-controlled randomized trials of longer duration are needed.

Continuous soy isoflavones exposure from weaning to maturity induces downregulation of ovarian steroidogenic factor 1 gene expression and corresponding changes in DNA methylation pattern

Female Wistar rats were treated with orally administered soy isoflavones at concentrations of 0, 25, 50, or 100mg/kg body weight from weaning until sexual maturity (3 mo.), and ovarian steroidogenesis was evaluated. After soy isoflavones were administered, a significant (P<0.05) decrease (44%) in the serum estrodial levels of the high-dose (HD) group were observed. Cultured granulosa cells from the middle- (MD) and HD groups showed significantly (P<0.05) reduced (31%, 45%, respectively) in vitro estradiol secretion, and those from the HD group showed significantly (P<0.05) reduced progesterone (25%) secretion. Compared with the control group, the mRNA expression of the steroidogenic acute regulatory protein (Star), cytochromeP450 cholesterol side chain cleavage (Cyp11a1 and Cyp19a1), and hydroxysteroid dehydrogenase 3b (Hsd3b) genes also decreased. Real-time quantitative PCR and Western blotting revealed a significant (P<0.05) decrease in key transcription factor steroidogenic factor-1 (SF-1) expression in the HD group. The detection of DNA methylation using bisulfitesequencing PCR (BSP) suggested a significantly (P<0.05) increased total methylation rate in the proximal SF-1 promoter in the HD group. Further studies showed that treatment with soy isoflavones can significantly (P<0.05) increase the mRNA expression of DNA methyltransferase (DNMT) 1 and DNMT3a. This study proved that soy isoflavone administration from weaning until sexual maturity could inhibit ovarian steroidogenesis, suggesting that SF-1 might play an important role in this effect. In addition, DNA methylation might play a role in the downregulation of SF-1 gene expression induced by soy isoflavones.

Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator associated with many biological processes. However, the roles and mechanisms of DNMT1 in skin aging are incompletely understood. Here we explored the role of DNMT1 in human skin fibroblasts senescence and its related regulatory mechanisms. DNMT1 expression decreased in passage-aged fibroblasts and DNMT1 silencing in young fibroblasts induced the senescence phenotype. MiR-217 is predicted to target DNMT1 mRNA and miR-217 expression increased in passage-aged fibroblasts. MiR-217 directly targeted the 3′-untranslated region (3′-UTR) of DNMT1 in HEK 293T cells and inhibited DNMT1 expression in fibroblasts. MiR-217 overexpression induced a senescence phenotype in young fibroblasts, and miR-217 downregulation in old HSFs partially reversed the senescence phenotype. However, these effects could be significantly rescued by regulating DNMT1 expression in fibroblasts. After regulating miR-217 levels, we analyzed changes in the promoter methylation levels of 24 senescent-associated genes, finding that 6 genes were significantly altered, and verified p16 and phosphorylated retinoblastoma (pRb) protein levels. Finally, an inverse correlation between DNMT1 and miR-217 expression was observed in skin tissues and different-aged fibroblasts. Together, these findings revealed that miR-217 promotes fibroblasts senescence by suppressing DNMT1-mediated methylation of p16 and pRb by targeting the DNMT1 3′-UTR.

Intracytoplasmic oxidative stress reverses epigenetic modifications in polycystic ovary syndrome.

In polycystic ovary syndrome (PCOS), substantial genetic and environmental alterations, along with hyperandrogenism, affect the quality of oocytes and decrease ovulation rates. To determine the mechanisms underlying these alterations caused specifically by an increase in plasma androgens, the present study was performed in experimentally-induced PCOS mice. As the study model, female B6D2F1 mice were treated with dehydroepiandrosterone (DHEA, 6mg per 100g bodyweight). After 20 days, oocytes at the germinal vesicle and metaphase II stages were retrieved from isolated ovaries and subsequent analyses of oocyte quality were performed for each mouse. DHEA treatment resulted in excessive abnormal morphology and decreased polar body extrusion rates in oocytes, and was associated with an increase in oxidative stress. Analysis of fluorescence intensity revealed a significant reduction of DNA methylation and dimethylation of histone H3 at lysine 9 (H3K9) in DHEA-treated oocytes, which was associated with increased acetylation of H4K12. Similarly, mRNA expression of DNA methyltransferase-1 and histone deacetylase-1 was significantly decreased in DHEA-treated mice. There was a significant correlation between excessive reactive oxygen species (ROS) production and increased histone acetylation, which is a novel finding and may provide new insights into the mechanism causing PCOS. The results of the present study indicate that epigenetic modifications of oocytes possibly affect the quality of maturation and ovulation rates in PCOS, and that the likely mechanism may be augmentation of intracytoplasmic ROS.

Elevated expression of DNMT1 is associated with increased expansion and proliferation of hematopoietic stem cells co-cultured with human MSCs.

BackgroundMesenchymal stem cells (MSCs) play an important role in hematopoietic stem cell (HSC) maintenance, proliferation, and apoptosis. DNA methyltransferase 1 (DNMT1) is considered an essential factor in the maintenance of HSCs in mammalian cells. Therefore, this study was conducted to evaluate the mRNA expression level of DNMT1 during cord blood (CB)-HSC ex vivo expansion with MSCs.MethodsEx vivo cultures of CB-HSCs were performed in three culture conditions for 7 days: cytokines, cytokines with MSCs, and only MSCs. Total and viable cell numbers were counted after 5 and 7 days using trypan blue stain, and the stem cell percentage was then evaluated by flow cytometry. Moreover, in vitro colony-forming unit assay was carried out to detect clonogenic potential of HSCs at days 0 and 7 using MethoCult H4434. Finally, DNMT1 mRNA expression level was evaluated by real-time polymerase chain reaction.ResultsMaximum CB-CD34+ cell expansion was observed on day 7 in all the three cultures. After 7 days, ex vivo expansion of CB-CD34+ cells indicated a significant decrease in DNMT1 expression in the cytokine cultures, whereas in the two co-culture conditions DNMT1 expression was increased. A significant difference between the number of CD34+ and CD34− cells in the cytokine co-culture system was observed.ConclusionThese data indicated that an elevated expression of DNMT1 is associated with increased expansion and proliferation of HSCs co-cultured with human MSCs. Hence, DNMT1 may be a potential factor in the maintenance of expanded HSCs co-cultured with human MSCs.

Effects and related mechanism of 5-aza-2'-deoxycytidine on endothelial function in rats with hyperhomocysteinemia

To investigate the effects and related mechanism of 5-aza-2'-deoxycytidine(Aza) on endothelial function in Hyperhomocysteinemia rats. Adult male SD rats were divided into 3 groups (n=7 each): control group, hyperhomocysteinemia (HHcy) group and Aza group according to the random number table. Control group rats were fed with normal diet. HHcy group rats were fed with diet adding 3% L-methionine. Aza group rats were fed with diet adding 3% L-methionine and Aza (0.5 mg/kg) injection for consecutive three days per week for 8 weeks. After 8 weeks, content of rat plasma homocysteine (Hcy) was detected by enzyme linked immunosorbent assay (ELISA). The rat mesenteric artery endothelium-dependent diastolic function was detected. The nitric oxide synthase (eNOS) activity and asymmetric dimethyl fine ammonia acid (ADMA) content were detected by ELISA, and the content of nitric oxide was detected by nitrate reductase method in the mesenteric arteries. The mRNA expression of DNA methyl transferase 1 (DNMT1) and dimethyl arginine acid dimethylamine hydrolase 2 (DDAH2) in the mesenteric arteries were detected by real-time fluorescence quantitative PCR, and the protein expressions of DNMT1 and DDAH2 in the mesenteric arteries were detected by Western blot. The DDAH2 promoter methylation level in the mesenteric arteries was detected by nested methylation specific PCR. (1) The content of plasma Hcy was significantly higher in the HHcy group and Aza group compared to the control group ((29.00±0.94) μmol/L and (26.43±0.47) μmol/L vs.(10.34±0.63) μmol/L, both P<0.01), which was significantly reduced in the Aza group compared with the HHcy group (P<0.05). (2) Acetylcholine-mediated relaxation at various concentrations was significantly lower in the HHcy group and the Aza group compared with the control group (both P<0.05), which was significantly increased in Aza group compared with HHcy group (P<0.05). SNP-mediated relaxation at various concentrations was similar among the three groups(all P>0.05). (3) Compared with the control group, the content of nitric oxide in the HHcy group was significantly decreased ((0.52±0.01) μmol/g vs.(0.42±0.00) μmol/g, P<0.01), which could be increased by Aza((0.49±0.01) μmol/g, P<0.05); the eNOS activity in the HHcy group was significantly decreased ((0.74±0.01) U/mg vs. (0.57±0.00) U/mg, P<0.01), which could be significantly increased by Aza ((0.65±0.01) U/mg, P<0.01); the content of ADMA in the HHcy group was significantly increased ((0.34±0.01) μmol/g vs. (0.37±0.00) μmol/g, P<0.05), which could be significantly decreased by Aza ((0.32±0.01) μmol/g, P<0.05). (4) Compared with the control group, the relative expression of DDHA2 mRNA in the HHcy group was significantly decreased (0.15±0.01 vs.0.12±0.01, P<0.01), which could be significantly increased by Aza (0.13±0.01, P<0.05); the relative expression of DDHA2 protein in the HHcy group was significantly decreased (0.31±0.02 vs. 0.24±0.01, P<0.01), which could be significantly increased by Aza (0.28±0.01, P<0.01). Compared with the control group, the relative expression of DNMT1 mRNA in the HHcy group was significantly increased (0.23±0.01 vs.0.43±0.01, P<0.01), which could be significantly decreased by Aza (0.39±0.01, P<0.05); the relative expression of DNMT1 protein in the HHcy group was significantly increased (0.35±0.01 vs. 0.50±0.01, P<0.01), which could be significantly decreased by Aza (0.47±0.01, P<0.05). (5) Compared with the control group, the methylated/non methylated ratio of DDHA2 promoter in the HHcy group was significantly increased (1.04±0.03 vs. 1.26±0.03, P<0.01), which could be significantly decreased by Aza (0.80±0.03, P<0.01). Aza can inhibit the activity of DNMT1, reduce DDAH2 promoter methylation level, increase the expression of DDAH2, decrease the content of ADMA, increase eNOS activity and content of nitric oxide, thus lead to the improvement of endothelial dysfunction in mesenteric artery of Hyperhomocysteinemia rats.

DNA Methyltransferase 1: A Potential Gene Therapy Target for Hepatocellular Carcinoma?

Background: DNA methyltransferase 1 (DNMT1) mutants display altered methylation patterns that may contribute to oncogenesis. We hypothesized that the silencing or inhibition of DNMT1 may affect the malignancy of hepatocellular carcinoma (HCC) cells. Methods: The HCC cell line KYN2 was used to construct 3 experimental groups: i) a DNMT1-siRNA group transfected with a green fluorescent protein (GFP) lentiviral vector to silence endogenous DNMT1 gene expression, which was confirmed by real-time quantitative polymerase chain reaction, ii) a 5-Aza-CdR group transfected with a null GFP lentiviral vector and treated with the DNMT1 inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR), and iii) a control group transfected with a null GFP lentiviral vector. Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy. Results: DNMT1 mRNA expression was significantly inhibited in DNMT1-silenced cells relative to control cells (p < 0.05), indicating successful transfection and gene expression knockdown. Cell proliferation was significantly inhibited in DNMT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). G1-to-S phase shifts were significantly increased in DNMT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). Apoptosis was significantly increased in DMNT1-siRNA and 5-Aza-CdR cells relative to control cells (p < 0.05). DMNT1-siRNA and 5-Aza-CdR cells displayed significantly reduced colony formation relative to control cells (p < 0.05). Notably, 5-Aza-CdR had more pronounced effects upon all these parameters than DNMT1 silencing. Conclusion: DNMT1 activity appears to positively contribute to the malignancy of HCC cells.

Associations of ChREBP and Global DNA Methylation with Genetic and Environmental Factors in Chinese Healthy Adults.

Age, gender, diet, gene and lifestyle have been reported to affect metabolic status and disease susceptibility through epigenetic pathway. But it remains indistinct that which factors account for certain epigenetic modifications. Our aim was to identify the influencing factors on inter-individual DNA methylation variations of carbohydrate response element binding protein (ChREBP) and global genome in peripheral blood leucocytes (PBLs). ChREBP DNA methylation was determined by bisulfite sequencing, and genomic 5mdC contents were quantified by capillary hydrophilic-interaction liquid chromatography/ in-source fragmentation/ tandem mass spectrometry system in about 300 healthy individuals. Eleven single nucleotide polymorphisms (SNPs) spanning ChREBP and DNA methyltransferase 1 (DNMT1) were genotyped by high resolution melting or PCR-restriction fragment length polymorphism. DNMT1 mRNA expression was analyzed by quantitative PCR. We found ChREBP DNA methylation levels were statistically associated with age (Beta (B) = 0.028, p = 0.006) and serum total cholesterol concentrations (TC) (B = 0.815, p = 0.010), independent of sex, concentrations of triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDL-C), fasting blood glucose and systolic blood pressure, diastolic blood pressure, PBLs counts and classifications. The DNMT1 haplotypes were related to ChREBP (odds ratio (OR) = 0.668, p = 0.029) and global (OR = 0.450, p = 0.015) DNA methylation as well as LDL-C, but not DNMT1 expression. However, only the relation to LDL-C was robust to correction for multiple testing (ORFDR = 1.593, pFDR = 0.013). These results indicated that the age and TC were independent influential factors of ChREBP methylation and DNMT1 variants could probably influence LDL-C to further modify ChREBP DNA methylation. Certainly, sequential comprehensive analysis of the interactions between genetic variants and blood lipid levels on ChREBP and global DNA methylation was required.

Abstract 571: Non-reversed Increase In Shear Stress Magnitude Enhances Global Endothelial Cell Dna Methylation And Limits Extent of Collateral Artery Growth Following Femoral Arterial Ligation

Femoral artery ligation (FAL) in the mouse leads to increased flow across all collateral arteries with some collateral segments experiencing increased flow and reversed flow direction. We have recently shown that these “reversed flow” segments exhibit remarkably permanent, amplified arteriogenesis compared to those exposed to “non-reversed” flow. As the role of epigenetics in arteriogenesis remains unknown, we sought to determine the effect of biomimetic shear stress waveforms on DNA methylation and DNA methyltransferase 1 (DNMT1) expression in endothelial cells. Next generation sequencing of bisulfite converted genomic DNA was used to generate global DNA methylation profiles of ECs exposed to shear stress waveforms biomimetic of our “reversed” and “non-reversed” conditions. We found that the “non-reversed” waveform increased gene promoter hypermethylation in-vitro. Furthermore, DNMT1 mRNA expression was increased 1.25-fold in non-reversed conditions compared to reversed conditions as determined by RT-PCR (p≤0.05, n=8). We then tested the hypothesis that increased DNMT1 expression and subsequent DNA hypermethylation, elicited by a “non-reversed” flow waveform, limits steady state, long-term arteriogenic capacity in-vivo. Whole mount vascular casting was used to determine the lumenal diameter of both reversed and non-reversed collateral artery segments from mice treated with 5-AZA, a preferential inhibitor of DNMT1, or vehicle control 14 days post-FAL for an additional 14 days. We found that lumenal diameter was significantly enhanced in non-reversed collateral segments of 5-AZA treated mice (70.9±7.9μm vs. 50.1±3.4μm, p≤0.05, n=6) while there was no difference in reversed segments. Together, we conclude that a non-reversed, increased shear stress magnitude results in a rapid increase DNMT1 expression and DNA hypermethylation, limiting the extent of arteriogenesis in non-reversed collateral artery segments of the mouse gracilis muscle. However, inhibition of DNMT1 with 5-AZA enables for enhanced collateral growth. Further interrogation of the central relationships between hemodynamics, epigenetics, and collateral artery growth may lead to durable therapeutic options for arterial occlusive diseases.

Expression of DNA methyltransferase mRNA in children with persistent and chronic immune thrombocytopenia

Objective To discuss DNA methylation's effect on pathogenesis of pediatric immune thrombocytopenia (ITP)through detecting the expression level of DNA methyltransferases (DNMTs)mRNA in peripheral blood lymphocytes of children with ITP. Methods Two mL peripheral blood was collected from each of 25 children with persistent and chronic ITP and 20 healthy children(the healthy control group)by using aseptic method in the pediatric ward of the First Affiliated Hospital of Xinxiang Medical University from January 2014 to January 2015.First ethylenediamine tetraacetic acid (EDTA) was used as the anticoagulant.Then separate the mononuclear cells, extract RNA and detect expression levels of DNMT1, DNMT3A and DNMT3B mRNA using reverse transcription-polymerase chain reaction (RT-PCR) method. Results (1)The blood platelet(PLT) of children with persistent and chronic ITP was (36.2±19.6)×109/L, which was obviously lower than the healthy control group(168.8±46.8)×109/L(t=-11.85, P=0.000). (2)The DNMT1 mRNA expression level of children with persistent and chronic ITP was 0.17±0.05, which was obviously lower than the healthy control group (0.27±0.10) (t=-3.912, P=0.001). The DNMT3A mRNA expression level of children with persistent and chronic ITP was 0.20±0.10, which was obviously lower than the healthy control group (0.32±0.11) (t=-3.779, P=0.000). The DNMT3B mRNA expression level of children with persistent and chronic ITP was 0.16±0.11, which was obviously lower than the healthy control group (0.31±0.11) (t=-4.641, P=0.000). (3) There was positive correlation between the expression of DNMT1 and DNMT3B mRNA(r=0.433, P=0.031). There was positive correlation between the expression of DNMT3A and DNMT3B mRNA(r=0.721, P=0.000). Conclusions (1) Children with persistent and chronic ITP have lower expression levels of DNMT1, DNMT3A, DNMT3B mRNA, which indicates that DNA methylation contributes to the pathogenesis of pediatric persistent and chronic ITP.(2) DNMTs have synergistic effect on DNA methylation of pediatric persistent and chronic ITP. Key words: Child; Immune thrombocytopenia, persistent, chronic; DNA methyltransferase

Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a crucial transcription factor that regulates the expression of defensive antioxidants and detoxification enzymes in cells. In a previous study, we showed that expression of the Nrf2 gene is regulated by an epigenetic modification. Rauvolfia verticillata, a traditional Chinese herbal medicine widely used in China, possesses anticancer and antioxidant effects. In this study, we investigated how Nrf2 is epigenetically regulated by reserpine, the main active component in R. verticillata, in mouse skin epidermal JB6 P+ cells. Reserpine induced ARE (antioxidant response element)-luciferase activity in HepG2-C8 cells. Accordingly, in JB6 P+ cells, it upregulated the mRNA and protein levels of Nrf2 and its downstream target genes heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), while it only increased the protein level of UDP-glucuronosyltransferase 1A1 (UGT1A1). Furthermore, reserpine decreased the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced colony formation of JB6 cells in a dose-dependent manner. DNA sequencing and methylated DNA immunoprecipitation further demonstrated the demethylation effect of reserpine on the first 15 CpGs of the Nrf2 promoter in JB6 P+ cells. Reserpine also reduced the mRNA and protein expression of DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferases 3a), and DNMT3b (DNA methyltransferases 3b). Moreover, reserpine induced Nrf2 expression via an epigenetic pathway in skin epidermal JB6 P+ cells, enhancing the protective antioxidant activity and decreasing TPA-induced cell transformation. These results suggest that reserpine exhibits a cancer preventive effect by reactivating Nrf2 and inducing the expression of target genes involved in cellular protection, potentially providing new insight into the chemoprevention of skin cancer using reserpine.

Prognostic value of the DNMTs mRNA expression and genetic polymorphisms on the clinical outcome in oral cancer patients.

Although the importance of the epigenetic changes in tumors, including oral squamous cell carcinomas (OSCCs), is now becoming apparent, the mechanisms that trigger or cause aberrant DNA methylation in cancer are still unrevealed. DNA methylation is regulated by a family of enzymes, DNA methyltransferases (DNMTs). DNMT gene expression analysis, as well as genetic polymorphisms, has not been previously evaluated in OSCC. In 65 OSCC patients, SYBR Green real-time PCR method was assessed for relative quantification of DNMT1, DNMT3A, and DNMT3B mRNAs, normalized to TATA-binding protein (TBP) mRNA. The expression levels of all three genes were dichotomized as high or low, with a twofold change of normalized mRNA expression used as the cutoff value. Polymorphisms in DNMT1 (rs2228612) and DNMT3B (rs406193) were analyzed in 99 OSCCs by TaqMan SNPs genotyping assays. DNMT1, DNMT3A, and DNMT3B were overexpressed in 36.9, 26, and 23% of the OSCC patients, respectively. DNMT1 overexpression was significantly associated with the overall survival, p=0.029, and relapse-free survival of OSCC patients, p=0.003. Patients with DNMT1 overexpression, as an independent prognostic factor, had a 2.385 times higher risk to relapse than those with lower expression. The DNMT1 A201G gene polymorphism was associated with a reduced overall survival in OSCC patients, p=0.036. Our results suggest that DNMT1 could play an important role in modulating OSCC patient survival. DNMT gene expression could be a potential prognostic marker that might lead to an improvement in diagnosis, prognosis, and prospective use of epigenetic-targeted therapy of OSCC.

Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen‐Induced Arthritis

Background. Wutou decoction (WTD) has been wildly applied in the treatment of rheumatoid arthritis and experimental arthritis in rats for many years. Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis; however, the effects of WTD on epigenetic changes are unclear. This study is set to explore the effects of WTD on DNA methylation and histone modifications in rats with collagen-induced arthritis (CIA). Methods. The CIA model was established by the stimulation of collagen and adjuvant. The knee synovium was stained with hematoxylin and eosin. The DNA methyltransferase 1 (DNMT1) and methylated CpG binding domain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR. The global DNA histone H3-K4/H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected. Results. Our data demonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (P < 0.05). The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (P < 0.05). Moreover, H3 acetylation of PBMCs was overexpressed in WTD compared with CIA (P < 0.05). Conclusions. WTD may modulate DNA methylation and histone modifications, functioning as anti-inflammatory potential.

The Effect of Schizandrol A-Induced DNA Methylation on SH-SY5YAB 1-40 Altered Neuronal Cell Line: A Potential Use in Alzheimer’s Disease

Disturbances in DNA methylation are postulated to result in various central nervous system diseases including Alzheimer’s disease (AD). The SH-SY5Y neuronal cell line treated with Aβ1-40 (5 μmol/L) protein is considered to be a model of AD. Hence the aim of this study was to examine the influence of Schizandrol A, a plant extract, on DNA methylation in SH-SY5Y cells exposed to Aβ1-40. Aβ1-40 were incubated with varying concentrations of Sehizandrol A to a final concentration of 1 (low), 3 (intermediate) or 9 μg/ml (high). Exposure of SH-SY5Y with Aβ1-40 reduced viability, and altered cellular morphology and mRNA expression of DNA methyltransferase (DNMT3A) and DNMT3B. Treatment with 1 or 3 μg/ml Sehizandrol A resulted in normal cell morphology as well as elevated cell number, enhanced viability, and increased mRNA expression of DNMT3A and DNMT3B compared to saline. However, an increase in Sehizandrol A to 9 μg/ml produced a fall in cell viability, as well as a decrease in mRNA DNMT3A and DNMT3B expression to control levels. Data demonstrated that Schizandrol A at 1 or 3 μg/ml improved cell morphological appearance and viability of Aβ1-40 injured SH-SY5Y cells by an enhanced DNA methylation pathway.

Association of DNA methyltransferases expression with global and gene-specific DNA methylation in colorectal cancer cells.

There are conflicting reports regarding the association between DNA methyltransferases (DNMTs) expression and global or gene-specific DNA methylation in colorectal cancer (CRC) cells. To correlate DNMTs expression with DNA methylation, we quantified DNMT1, DNMT3A and DNMT3B mRNA levels in five CRC cell lines (HCT116, LS180, HT29/219, Caco2 and SW742) by real-time reverse-transcriptase polymerase chain reaction (PCR) assay. In addition, we examined the global 5-methyl cytosine levels and the methylation patterns of 12 CpG islands in these CRC cells by enzyme-linked immunosorbent assay and methylation-specific PCR methods, respectively. The average expression levels of three DNMTs in HCT116, Caco2, HT29/219 and SW742, relative to the expression level in LS180 (taken to be 1), were 90.1, 31.6, 2.66 and 1.86. Our data indicated that overall about 1.45%, 1.03%, 0.98%, 0.86% and 0.85% of the cytosines were methylated in the genome of HCT116, Caco2, HT29/219, SW742 and LS180 cells, respectively. The 5-mC percentages were positively correlated with the relative cellular DNMTs expression in five CRC cell lines as verified by Pearson correlation test. However, we found no positive correlation between mRNA expression of DNMTs and gene promoter hypermethylation in these cells. Our results suggest that cellular DNMT expression is positively correlated with global DNA methylation level but not with regional DNA hypermethylation at each locus.

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto‑oncogenes. The methylation expression rate of C‑myc was 87.5% in nasal tumor tissues and 15% in normal nasal epithelial tissues. The methylation expression rate of CEBPA was 100% in nasal tumor tissues and 40% in normal nasal epithelial tissues. The methylation expression rate of the EGFR gene was ~80% in the two tissues. In summary, the present study identified abnormal methylation of the C‑myc, CEBPA, GADD45G and THBS1 genes in nasal tumor tissues. The expression levels of DNMT1, C‑myc and CEBPA were upregulated and the expression of P53 and THBSI were downregulated in nasal tumor tissues, with a significant difference between the two groups (P<0.05). Therefore, it is suggested that these six genes may be used as diagnostic marker candidates for ENT. The results may serve as a foundation for screening of tumor‑specific markers for early diagnosis of ENT and further investigate the epigenetic mechanisms of enzootic nasal tumor virus (ENTV)‑induced nasal epithelium cell carcinoma.