mRNA Levels Of DNA Methyltransferases Research Articles

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

Maternal obesity alters methylation level of cytosine in CpG island for epigenetic inheritance in fetal umbilical cord blood

BackgroundOver the past few decades, global maternal obesity prevalence has rapidly increased. This condition may induce long-lasting pathophysiological effects on either fetal or infant health that could be attributable to unknown unique changes in the umbilical blood composition.MethodsA total of 34 overweight/obese and 32 normal-weight pregnant women were recruited. Fifteen umbilical blood samples including 8 overweight/obese subjects and 7 normal weight women were sequenced using Targeted Bisulfite Sequencing technology to detect the average methylation level of cytosine and identify the differentially methylated region (DMR). GO and KEGG analyses were then employed to perform pathway enrichment analysis of DMR-related genes and promoters. Moreover, the mRNA levels of methylation-related genes histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) were characterized in the samples obtained from these two groups.ResultsAverage methylated cytosine levels in both the CpG islands (CGI) and promoter significantly decreased in overweight/obese groups. A total of 1669 DMRs exhibited differences in their DNA methylation status between the overweight/obese and control groups. GO and KEGG analyses revealed that DMR-related genes and promoters were enriched in the metabolism, cancer and cardiomyopathy signaling pathways. Furthermore, the HDACs and DNMTs mRNA levels trended to decline in overweight/obese groups.ConclusionsDecreased methylated cytosine levels in overweight/obese women induce the gene expression activity at a higher level than in the control group. DMRs between these two groups in the fetal blood may contribute to the changes in gene transcription that underlie the increased risk of metabolic disorders, cancers and cardiomyopathy in their offspring.

Parental diuron exposure causes lower hatchability and abnormal ovarian development in offspring of medaka (Oryzias melastigma).

Diuron is one of the most widely used herbicides worldwide. It has been widely detected in various aquatic environments, especially in marine ecosystems. Although direct effects of diuron exposure on various organisms have been reported, little is known about its effects on marine fishes including multigenerational effects. Herein, the filial generation (F1) of diuron-exposed marine medaka (Oryzias melastigma) (F0) was raised in clean seawater from fertilized eggs to adulthood and used as a marine fish model to study the potential multigenerational effects of diuron. We found that the successful hatching of F1 larvae was significantly reduced and that ovarian development in F1 females was retarded. A significant increase in the percentage of previtellogenic oocytes, along with a visual decrease in the percentage of vitellogenic and mature oocytes in the F1 ovary, were observed. The hormone levels of the hypothalamus-pituitary-gonad-liver axis and vitellogenin-related transcription were downregulated. In addition, the mRNA levels of DNA methyltransferase in the brain, ovary and liver of F1 adult fish exhibited significant upregulation, suggesting that the probable underlying multigenerational mechanism might be associated with epigenetic modifications. Taken together, these results demonstrated that chronic environmental diuron exposure in F0 marine medaka can inhibit F1 ovary development and suggested that diuron may affect marine fish thriving in the ocean.

MRNA Expressions of Methylation Related Enzymes and Duration of Thermal Conditioning in Chicks.

DNA methylation regulates gene expression by modifying the nucleosome structure of DNA, without altering the gene sequence. It has been reported that DNA methylation reactions are catalyzed by several enzymes. In chickens, thermal conditioning treatment affects the central DNA methylation levels. The purpose of this study was to clarify the changes in DNA methylation and demethylation factors during thermal conditioning in the hypothalamus of 3-day-old chicks. Male chicks (3-days old) were exposed to 40±0.5°C as a thermal conditioning treatment for 1, 2, 6, 9, or 12 h. The control chicks were kept in a thermoneutral zone (30±0.2°C). After thermal conditioning, the mRNA levels of DNA methyltransferase (DNMT)-1, -3a, -3b, and ten-eleven translocation (TET)-1, -2, and -3 in the hypothalamus were measured by q-PCR. The mRNA levels of DNMT-3a and TET-1 were increased by thermal conditioning. Moreover, the expression level of TET-1 increased with the loading time of the thermal conditioning. The gene expressions of DNMT-1, DNMT-3b, TET-2, and TET-3 were not affected by thermal conditioning. Since DNMT-3a is a catalyst for de-novo DNA methylation and TET-1 catalyzes the oxidation of methylated cytosine, it is suggested that the thermal conditioning increased the activation of DNA methylation and demethylation factors, which occur in the hypothalamus of neonatal chicks.

Oxytocin prevents cue-induced reinstatement of oxycodone seeking: Involvement of DNA methylation in the hippocampus.

Oxycodone is one of the most commonly used analgesics in the clinic. However, long-term use can contribute to drug dependence. Accumulating evidence of changes in DNA methylation after opioid relapse has provided insight into mechanisms underlying drug-associated memory. The neuropeptide oxytocin is reported to be a potential treatment for addiction. The present study sought to identify changes in global and synaptic gene methylation after cue-induced reinstatement of oxycodone conditioned place preference (CPP) and the effect of oxytocin. We analyzed hippocampal mRNA of synaptic genes and also synaptic density in response to oxycodone CPP. We determined the mRNA levels of DNA methyltransferases (Dnmts) and ten-eleven translocations (Tets), observed global 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels, and measured DNA methylation status of four synaptic genes implicated in learning and memory (Arc, Dlg1, Dlg4, and Syn1). Both synaptic density and the transcription of 15 hippocampal synaptic genes significantly increased following cue-induced reinstatement of oxycodone CPP. Oxycodone relapse was also related to markedly decreased 5-mC levels and decreased transcription of Dnmt1, Dnmt3a, and Dnmt3b; in contrast, 5-hmC levels and the transcription of Tet1 and Tet3 were increased. Oxycodone exposure induced DNA hypomethylation at the exons of the Arc, Dlg1, Dlg4, and Syn1 genes. Intracerebroventricular (ICV) administration of oxytocin (2.5μg/μl) specifically blocked oxycodone relapse, possibly by inhibition of Arc, Dlg1, Dlg4, and Syn1 hypomethylation in oxycodone-treated rats. Together, these data indicate the occurrence of epigenetic changes in the hippocampus following oxycodone relapse and the potential role of oxytocin in oxycodone addiction.

DNA Methyltransferases as Potential Biomarkers for HCV Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a major health concern in many countries including Egypt. The alteration in DNA methylation that was observed in HCC patients suggests a possible role of DNA methyltransferases (DNMTs) in the disease pathogenesis in addition to potential role as a disease biomarker. To study the change in DNMTs expression in chronic HCV infected patients as potential non- invasive biomarker for diagnosis of hepatocellular carcinoma. 26 patients with HCC, 45 patients with liver cirrhosis, 20 chronic HCV patients and 20 apparently healthy individuals as a control group were enrolled in this study. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was performed for all study participants. A significant difference in DNMTs expression was observed among the studied groups. Receiver operating characteristics (ROC) curve analysis revealed that with a cutoff value of 3.16 for DNMT 3A expression, sensitivity and specificity were 80.8 and 95.6% respectively and area under curve (AUC) was 0.958, p < 0.001 for discriminating hepatocellular carcinoma among post hepatitis C cirrhotic patients. Besides DNMT 3B relative expression cutoff value of 3.10 showed 84.6% sensitivity and 77.8% specificity and AUC was 0.888, p < 0.001. On the other hand, cutoff value 0.65 for DNMT1 relative expression showed 92.3% sensitivity and 44.4% specificity and AUC was 0.72, p= 0.002. DNMT1, DNMT 3A and DNMT 3B have significant positive correlation with the level of AFP (p-value = 0.003, 0.004 and 0.008 respectively). The relative expression of DNMT3B was significantly correlated to focal lesion size (p-value = 0.015). High DNMTs expression was significantly associated with the presence of multiple focal lesions but not with the Child Pugh grade (p> 0.05). The mRNA levels of DNMTs could be a potential biomarker for early detection of HCC development.<br />.

Abstract MP04: High Fat Diet Exposure Induces Epigenetic Modulation In The Brain Renin Angiotensin System

Cardiometabolic Diseases (CMD) are one of the highest causes of morbidity and mortality worldwide. In-utero, environmental insults can program brain’s progeny through changes in gene expression level affecting cardiometabolic functions. Methylation and demethylation of DNA take place by DNA methyltransferase (DNMT) and Ten eleven translocation (TET) enzymes, respectively. Expression of renin angiotensin system (RAS) on pre-autonomic neurons in the hypothalamus is believed to play a pivotal role in sympathetic activity controlling metabolic and blood pressure (BP) homeostasis. We hypothesize that perinatal exposure to high fat high sucrose diet (HFHSD) programs the brain RAS through methylation mechanisms that lead to autonomic dysfunction and hypersensitivity to CMD. C57BL6/J dams were fed HFHSD or regular diet (RD) and mated with RD sires. After weaning, offspring were raised on RD. Three-month old progeny underwent 24 h BP recording (telemetry), measurement of fasted blood glucose (FBG), assessment of autonomic function (pharmacological) and baroreflex sensitivity (sequence method). Mice were then sacrificed, and brains harvested for quantification of mRNA levels of DNMT, TET and RAS components in the hypothalamus. HFHSD-exposed male offspring showed elevation in systolic BP (140.2 ±1.9 vs. 131.2 ±2.4 mmHg, p=0.006), heart rate (611.3 ±7.8 vs 562.5 ±7 bpm, p&lt;0.0001) and FBG (172.7 ±6.3 vs. 142.6 ±6.3 mg/dl, p=0.018) but not females. Cardiac sympathetic activity was higher in HFHSD-exposed mice (-148 ±11.5 vs. -35.2 ±4.7 bpm, p&lt;0.05). Surprisingly, sympathetic vascular tone was only increased in HFHSD-exposed females (-64.6 ±5.7 vs. -36.7 ±2.9 mmHg, p&lt;0.05) while spontaneous baroreflex gain was improved in HFHSD-exposed males (5.3 ±0.7 vs. 2.6 ±0.4 msec/mmHg, p&lt;0.05). In the brain, DNMT1, DNMT3a and TET1, 2 and 3 mRNA expression were downregulated (p&lt;0.05) while AT 1a R and ACE2 genes were upregulated (p=0.02) in HFHSD-exposed mice. We conclude that perinatal exposure to a hypercaloric diet epigenetically programs the progeny’s brain through methylation leading to a pre-hypertensive and pre-diabetic phenotype associated with changes in the expression of RAS genes which could eventually result in increased susceptibility to CMD.

Prenatal Exposure to Bisphenol A, E, and S Induces Transgenerational Effects on Male Reproductive Functions in Mice.

This study was performed to examine the transgenerational effects of bisphenol (BP) A analogs, BPE, and BPS on male reproductive functions using mice as a model. CD-1 mice (F0) were orally exposed to control treatment (corn oil), BPA, BPE, or BPS (0.5 or 50 µg/kg/day) from gestational day 7 (the presence of vaginal plug = 1) to birth. Mice from F1 and F2 offspring were used to generate F3 males. Prenatal exposure to BPA, BPE, and BPS decreased sperm counts and/or motility and disrupted the progression of germ cell development as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F3 males. Dysregulated serum levels of estradiol-17β and testosterone, as well as expression of steroidogenic enzymes in F3 adult testis were also observed. In the neonatal testis, although apoptosis and DNA damage were not affected, mRNA levels of DNA methyltransferases, histone methyltransferases, and their associated factors were increased by BP exposure. Furthermore, BP exposure induced immunoreactive expression of DNMT3A in Sertoli cells, strengthened DNMT3B, and weakened H3K9me2 and H3K9me3 in germ cells of the neonatal testis, whereas DNMT1, H3K4me3, and H3K27ac were not affected. In adult testis, stage-specific DNMT3B was altered by BP exposure, although DNMT3A, H3K9me2, and H3K9me3 expression remained stable. These results suggest that prenatal exposure to BPA, BPE, and BPS induces transgenerational effects on male reproductive functions probably due to altered epigenetic modification following disruption of DNMTs and histone marks in the neonatal and/or adult testis.

Reversal of oxycodone conditioned place preference by oxytocin: Promoting global DNA methylation in the hippocampus

Repeated exposure to the opioid agonist, oxycodone, can lead to addiction. Accumulating evidence has shown that oxytocin (OT), a neurohypophyseal neuropeptide, could reduce the abuse potential of drugs. Recent studies suggest that epigenetic regulation through DNA methylation are involved in neuroadaptations. The current study was conducted to investigate the effects of OT on oxycodone conditioned place preference (CPP) and the epigenetic mechanism of OT in the hippocampus. For induction of CPP, oxycodone (3.0 mg/kg, i. p.) was administrated to male Sprague-Dawley rats once every other day during an eight-day conditioning phase. Global 5-methylcytosine (5-mC) level was determined based on CPP procedure, including acquisition, expression, extinction and reinstatement. We also measured mRNA levels of DNA methyltransferases (Dnmts), ten-eleven translocations (Tets) and synaptic genes (Psd95, Shank2, Gap43, etc.), and determined synaptic density after restraint stress-induced reinstatement of oxycodone CPP. The results showed that OT (2.5 μg, i. c.v.) pretreatment specifically inhibited the CPP acquisition and expression, facilitated the CPP extinction, and abolished restraint stress-induced reinstatement of oxycodone CPP. OT markedly inhibited global 5-mC changes induced by oxycodone CPP in the four phases. Following restraint stress-induced reinstatement of oxycodone CPP, OT significantly increased mRNA levels of Dnmt1, decreased Tet1 mRNA, synaptic proteins and synaptic density in the hippocampus. Our study indicated that reversal of global DNA hypomethylation through OT could significantly attenuate the rewarding effects induced by oxycodone. Our results suggested that OT could be specific manipulation on oxycodone addiction.

Improves the In Vitro Developmental Competence and Reprogramming Efficiency of Cloned Bovine Embryos by Additional Complimentary Cytoplasm.

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker® Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.

Circulating cell-free DNA of methylated insulin-like growth factor-binding protein 7 predicts a poor prognosis in hepatitis B virus-associated hepatocellular carcinoma after hepatectomy

The initiation and progression of hepatocellular carcinoma (HCC) is a multistage process involving a variety of changes at the gene level. Methylation of insulin-like growth factor-binding protein 7 (IGFBP7) plays a crucial role in HCC development. The main purpose of this study was to investigate the relationship between oxidative stress, DNA methyltransferases (DNMTs) expression, and IGFBP7 methylation, and to evaluate the prognostic value of serum IGFBP7 methylation status in patients with HCC after hepatectomy. We enrolled 155 patients with HCC undergoing surgical resection. The IGFBP7 methylation status, DNMTs mRNA levels and malondialdehyde (MDA), xanthine oxidase (XOD), reduced glutathione hormone (GSH), and glutathione-S-transferases (GST) levels were detected. MDA and XOD levels were significantly higher in IGFBP7 methylated group than unmethylated group, while GSH level was lower in methylated group than unmethylated group. The DNMT1 and DNMT3a mRNA levels were higher in IGFBP7 methylated group than unmethylated group. Kaplan–Meier curve analysis revealed that IGFBP7 promoter methylation was significantly correlated with overall survival (OS) (p < .001). Moreover, IGFBP7 methylation was an independent prognostic predictor for OS (p = .000) and early tumour recurrence (ETR) (p = .008) in HCC after hepatectomy. Our results indicated that IGFBP7 promoter methylation was associated with oxidative stress and DNMTs expression. Meanwhile, IGFBP7 promoter methylation was associated with OS and ETR, indicating that it might serve as a potentially independent prognostic factor in patients with HCC after hepatectomy.

Alterations of Global DNA Methylation and DNA Methyltransferase Expression in T and B Lymphocytes from Patients with Newly Diagnosed Autoimmune Thyroid Diseases After Treatment: A Follow-Up Study.

Dysregulated DNA methylation in lymphocytes has been linked to autoimmune disorders. The aims of this study were to identify global DNA methylation patterns in patients with autoimmune thyroid diseases and to observe methylation changes after treatment for these conditions. A cross-sectional study was conducted, including the following patients: 51 with newly diagnosed Graves' disease (GD), 28 with autoimmune hypothyroidism (AIT), 29 with positive thyroid autoantibodies, and 39 matched healthy volunteers. Forty GD patients treated with radioiodine or antithyroid drugs and 28 AIT patients treated with L-thyroxine were followed for three months. Serum free triiodothyronine, free thyroxine, thyrotropin, thyroid peroxidase antibodies, thyroglobulin antibodies, and thyrotropin receptor antibodies were assayed using electrochemiluminescent immunoassays. CD3+ T and CD19+ B cells were separated by flow cytometry for total DNA and RNA extraction. Global DNA methylation levels were determined by absorptiometry using a methylation quantification kit. DNA methyltransferase (DNMT) expression levels were detected by real-time polymerase chain reaction. Hypomethylation and down-regulated DNMT1 expression in T and B lymphocytes were observed in the newly diagnosed GD patients. Neither the AIT patients nor the positive thyroid autoantibodies patients exhibited differences in their global DNA methylation status or DNMT mRNA levels compared with healthy controls. Antithyroid drugs restored global methylation and DNMT1 expression in both T and B lymphocytes, whereas radioiodine therapy affected only T cells. L-thyroxine replacement did not alter the methylation or DNMT expression levels in lymphocytes. The global methylation levels of B cells were negatively correlated with the serum thyroid peroxidase antibodies in patients with autoimmune thyroid diseases. Hyperthyroid patients with newly diagnosed GD had global hypomethylation and lower DNMT1 expression in T and B lymphocytes. The results provide the first demonstration that antithyroid drugs or radioiodine treatment restore global DNA methylation and DNMT1 expression with concurrent relief of hyperthyroidism.

38 Improved Cloning Efficiency and Developmental Potential in Bovine Somatic Cell Nuclear Transfer with the New Technology

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by IVF; however, its efficiency remains low. In this study, we examined the effects of cytoplasm restoration of enucleated oocyte, by injecting ~30% of the cytoplasm of a donor oocyte to restore the enucleated oocyte cytoplasm volume to normal, on the developmental competence and quality of bovine cloned embryos during pre-implantation using the TUNEL assay, quantitative reverse transcription PCR (RT-qPCR) and immunocytochemistry. The experiment was conducted in 6 replicates. The differences in embryo development and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. The level of statistical significance was set at P &lt; 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (P &lt; 0.05) in the cytoplasmic injected group than in the traditional SCNT group (61.5 ± 1.3% v. 39.7 ± 2.1% and 28.9 ± 0.8% v. 20.2 ± 1.3%, respectively). Furthermore, the beneficial effects of cytoplasmic injection on the cloned embryos were associated with a significantly increased (P &lt; 0.05) total cell number in Day 8 blastocysts compared with the traditional SCNT group (176.2 ± 6.5 v. 119.3 ± 7.7; P &lt; 0.05); however, there was no difference (P &gt; 0.05) between the number of apoptotic cells per blastocyst in the cytoplasmic injected group and in the traditional SCNT group (3.5 ± 1.1 v. 4.1 ± 0.8). Moreover, cytoplasm restoration of enucleated oocyte significantly increased (P &lt; 0.05) mitochondrial activity, as identified by MitoTracker Green (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription-qPCR showed that the mRNA levels of DNA methyl-transferase 1 and DNA methyl-transferase 3a were significantly decreased (P &lt; 0.05) in cytoplasmic injected group compared with the traditional SCNT group, but did not significantly differ (P &gt; 0.05) between the cytoplasmic injected and IVF groups. Taken together, these data suggest that cytoplasm restoration of enucleated oocyte improves in vitro developmental competence and quality of bovine cloned embryos, as evidenced by increased total cell numbers, reprogramming efficiency, and mitochondria activity. This work was partly supported by grant from the Next-Generation BioGreen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co. Felix Pets) and BK21plus.

DNA methylation in demyelinated multiple sclerosis hippocampus

Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of the human central nervous system (CNS). Memory impairments and hippocampal demyelination are common features in MS patients. Our previous data have shown that demyelination alters neuronal gene expression in the hippocampus. DNA methylation is a common epigenetic modifier of gene expression. In this study, we investigated whether DNA methylation is altered in MS hippocampus following demyelination. Our results show that mRNA levels of DNA methyltransferase were increased in demyelinated MS hippocampus, while de-methylation enzymes were decreased. Comparative methylation profiling identify hypo-methylation within upstream sequences of 6 genes and hyper-methylation of 10 genes in demyelinated MS hippocampus. Genes identified in the current study were also validated in an independent microarray dataset generated from MS hippocampus. Independent validation using RT-PCR revealed that DNA methylation inversely correlated with mRNA levels of the candidate genes. Queries across cell-specific databases revealed that a majority of the candidate genes are expressed by astrocytes and neurons in mouse and human CNS. Taken together, our results expands the list of genes previously identified in MS hippocampus and establish DNA methylation as a mechanism of altered gene expression in MS hippocampus.

Brain-Derived Neurotrophic Factor Mediated Perfluorooctane Sulfonate Induced-Neurotoxicity via Epigenetics Regulation in SK-N-SH Cells.

Perfluorooctane sulfonate (PFOS), a new kind of persistent organic pollutant, is widely distributed in the environment and exists in various organisms, where it is also a neurotoxic compound. However, the potential mechanism of its neurotoxicity is still unclear. To examine the role of epigenetics in the neurotoxicity induced by PFOS, SK-N-SH cells were treated with different concentrations of PFOS or control medium (0.1% DMSO) for 48 h. The mRNA levels of DNA methyltransferases (DNMTs) and Brain-derived neurotrophic factor (BDNF), microRNA-16, microRNA-22, and microRNA-30a-5p were detected by Quantitative PCR (QPCR). Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the protein levels of BDNF, and a western blot was applied to analyze the protein levels of DNMTs. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the BDNF promoter I and IV. Results of MTT assays indicated that treatment with PFOS could lead to a significant decrease of cell viability, and the treated cells became shrunk. In addition, PFOS exposure decreased the expression of BDNF at mRNA and protein levels, increased the expression of microRNA-16, microRNA-22, microRNA-30a-5p, and decreased the expression of DNMT1 at mRNA and protein levels, but increased the expression of DNMT3b at mRNA and protein levels. Our results also demonstrate that PFOS exposure changes the methylation status of BDNF promoter I and IV. The findings of the present study suggest that methylation regulation of BDNF gene promoter and increases of BDNF-related-microRNA might underlie the mechanisms of PFOS-induced neurotoxicity.

Intra-uterine undernutrition amplifies age-associated glucose intolerance in pigs via altered DNA methylation at muscle GLUT4 promoter.

The present study aimed to investigate the effect of maternal malnutrition on offspring glucose tolerance and the epigenetic mechanisms involved. In total, twelve primiparous Landrace×Yorkshire gilts were fed rations providing either 100 % (control (CON)) or 75 % (undernutrition (UN)) nutritional requirements according to the National Research Council recommendations, throughout gestation. Muscle samples of offspring were collected at birth (dpn1), weaning (dpn28) and adulthood (dpn189). Compared with CON pigs, UN pigs showed lower serum glucose concentrations at birth, but showed higher serum glucose and insulin concentrations as well as increased area under the blood glucose curve during intravenous glucose tolerance test at dpn189 (P<0·05). Compared with CON pigs, GLUT-4 gene and protein expressions were decreased at dpn1 and dpn189 in the muscle of UN pigs, which was accompanied by increased methylation at the GLUT4 promoter (P<0·05). These alterations in methylation concurred with increased mRNA levels of DNA methyltransferase (DNMT) 1 at dpn1 and dpn28, DNMT3a at dpn189 and DNMT3b at dpn1 in UN pigs compared with CON pigs (P<0·05). Interestingly, although the average methylation levels at the muscle GLUT4 promoter were decreased at dpn189 compared with dpn1 in pigs exposed to a poor maternal diet (P<0·05), the methylation differences in individual CpG sites were more pronounced with age. Our results indicate that in utero undernutrition persists to silence muscle GLUT4 likely through DNA methylation during the ageing process, which may lead to the amplification of age-associated glucose intolerance.

Methylation of the Glutathione-S-Transferase P1 Gene Promoter Is Associated with Oxidative Stress in Patients with Chronic Hepatitis B.

Glutathione-S-transferase P1 (GSTP1) and glutathione-S-transferase M3 (GSTM3) catalyze the glutathione-related clearance of xenobiotics. The methylation of these gene promoters was associated with oxidative stress that induced liver damage. This study aims to explore the relationship among GSTP1 and GSTM3 methylation, DNA methyltransferases (DNMTs) expression, and oxidative stress in patients with chronic hepatitis B (CHB). We retrospectively enrolled 153 patients with CHB and 40 healthy controls (HCs). The GSTP1 and GSTM3 methylation status, DNMTs mRNA levels in peripheral mononuclear cells (PBMCs) and TNF-α and malondialdehyde (MDA) levels in plasma were detected. GSTP1 methylation was significantly higher in patients with CHB than HCs (P = 0.047). Patients with HBeAg-positive CHB showed significantly higher GSTP1 methylation than those with HBeAg-negative CHB (P = 0.017) and HCs (P = 0.007). No significant difference was observed between GSTP1 methylation in HBeAg-negative CHB and HCs (P = 0.191). DNMT1 and DNMT3a mRNA levels were significantly higher in participants with GSTP1 methylation than those without. In patients with CHB, the degree of GSTP1 promoter methylation was significantly correlated with DNMT1 mRNA, DNMT3a mRNA, TNF-α, MDA, HBeAg, ALT, AST and TBIL. In contrast, no significant difference was found between GSTM3 methylation in patients with CHB and HCs (P = 0.079). Meanwhile, no significant difference could be observed between GSTM3 promoter methylation in patients with HBeAg-positive CHB and HBeAg-negative CHB (P = 0.146). Therefore, this study demonstrated that GSTP1 hypermethylation was associated with DNMT1, DNMT3a overexpression and oxidative stress in patients with HBeAg-positive CHB.

Abnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats

Pathogenesis of neuropathic pain is complex and not clearly understood. Glutamate decarboxylase 67 (GAD 67) is a key synthetic enzyme for the main inhibitory transmitter gamma-aminobutyric acid (GABA), and diminishes in the spinal dorsal horn in rats following chronic constriction injury (CCI). GAD 67 is coded by gene GAD 1. DNA methylation can regulate the expression of GAD 67 by regulating the methylation of GAD 1 promoter in the psychotic brain. DNA methylation is primarily mediated by DNA methyltransferases (DNMTs) and methyl-DNA binding domain proteins (MBDs). In this study, in order to discover whether DNA methylation regulates GAD 67 expression in the spinal cord in CCI rats and is involved in neuropathic pain, we examined mRNA levels of DNMTs, MBDs and GAD 67 with real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), and methylation of GAD 1 promoter with Pyromark CpG Assays in the lumbar spinal cord in CCI rats on day 14 after surgery. Our results showed that DNMT3a, DNMT3b and methyl-CpG binding protein 2 (MeCP2) expression increased, MBD2 expression decreased, and DNMT1, MBD1 and MBD3 expression hardly changed in the lumbar spinal cord in CCI rats on day 14 after surgery. GAD 67 expression decreased, and methylation of GAD 1 promoter increased in the lumbar spinal cord in CCI rats on day 14 after surgery. These results indicate that decreased GAD 67 may be associated with increased GAD 1 promoter methylation, which may be mediated by DNMT3a, DNMT3b, MeCP2 and MBD2 in CCI rats. These indicate that abnormal DNA methylation may be highly involved in CCI-induced neuropathic pain.

Corticosterone induces DNA methyltransferases expression in rat cortical neurons

Corticosterone (CORT) is the main glucocorticoid hormone involved in stress responses in rodents. It is established that CORT exerts its effects via glucocorticoid receptor (GR) and mineralocorticoid receptor that regulate downstream gene expression during development and adulthood. In our previous study, we have shown that maternal separation on postnatal day 15 increases DNA methyltransferase (DNMT) 1, 3A and 3B expression levels in rat nucleus accumbens lasting into adulthood (Anier et al. 2014). However, the exact mechanism how maternal separation alters DNMT expression is unclear. We hypothesize that stress-induced GR stimulation may increase the expression levels of DNMTs and alter long-term DNA methylation-demethylation balance in infant rat brain. Our aim is to evaluate the effect of CORT and maternal separation on the expression levels of DNMTs in rat cortex. In rat primary cortical neurons, CORT treatment increased mRNA levels of DNMT3A and DNMT3B. GR antagonist mifepristone significantly decreased CORT-induced DNMTs mRNA levels indicating GR stimulation-dependent upregulation of DNMTs expression. Higher mRNA levels of DNMT1, DNMT3A and DNMT3B in rat cortex at postnatal day 15 and increased plasma CORT levels suggest that elevated CORT upregulates DNMTs expression. Our results indicated that DNMTs are downstream targets of GR-dependent CORT stimulation and early life stress may induce aberrant DNA methylation pattern that could facilitate long-term changes in gene expression.

Tetrandrine affects the expression of DNMT in HL-60 cells

Objective To investigate the expression of DNA methyltransferase (DNMT) in HL-60 cells induced by tetrandrine (Tet). Methods HL-60 cells were treated with different concentrations of Tet and decitabine (DAC) alone and in combination with both. Methyl thiazolyl tetrazolium (MTT) assay was used to assess cytotoxic effect. Flow cytometry (FCM) was used to determine apoptosis rate. Real-time quantitative polymerase chain reaction (PCR) assay was used to quantify mRNA levels of DNMT. Western blot was used to quantify the expression of DNMT protein. Results Tet inhibited the growth and proliferation of HL-60 cells in a time- and dose-dependent manners (both P<0.01). Tet treated HL-60 cells after 48 h at the concentration of 2 μmol/L, and 4 μmol/L, the levels of DNMT gene and protein in the drug administration group decreased compared to the control group. After incubation for 48 h with Tet 2 μmol/L combined with DAC 4 μmol/L, the combination group was significantly depressed. Conclusions Tet could potently inhibit the growth and proliferation of HL-60 cells, reduce the expression levels of DNMT mRNA and protein, and have a more obvious effect in the combination group. Key words: TETRANDRINE/AD; HL-60 cells/DE/ME; DNA(cytosine-5-)-methyltransferase/ME