DNA Methyltransferase 1 mRNA Research Articles

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.

Expression and Clinical Significance of DNMT in Patients with Chronic Myeloid Leukemia

To investigate the expression and clinical significance of DNA methyltransferases (DNMT) mRNA in patients with chronic myeloid leukemia (CML). The expression levels of DNMT mRNA in mononucllear cells (MNC) of bone marrow or in peripheral blood of 93 CML patients in 3 different phases and 10 normal controls (NC) were detected by SYBR Green flurescent quatitative PCR. The relative expression levels of DNMT1 mRNA in NC, chronic phase CML (CML-CP), accelerated phase (CML-AP) and blastic phase (CML-BP) were 1.45 ± 0.22, 1.83 ± 0.63, 2.95 ± 0.87 and 3.24 ± 1.39 resectively. The expression of DNMT1 mRNA showed no statistically significant difference between CML-CP and NC (P = 0.28). The expression of DNMT1 mRNA in advanced stages (including CML-AP and CML-BP) of CML obviously increased in comparison with CML-CP and NC (P < 0.05). The expression of DNMT1 mRNA in CML-AP was not significantly different from that in CML-BP (P = 0.336). The relative expression levels of DNMT3a mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.29 ± 0.34, 1.34 ± 0.46, 2.33 ± 1.05 and 3.18 ± 1.23 resectively. And the expression levels of DNMT3a mRNA were not statistically significantly different between CML-CP and NC (P = 0.844). The results showed that the expression of DNMT3a mRNA in the advanced phase of CML significantly increased in comparison with that in CML-CP and NC (P < 0.05). Meanwhile, the expression of DNMT3a mRNA in CML-AP was not different from that in CML-BP (P = 0.304). The relative expression levels of DNMT3b mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.37 ± 0.31, 16.41 ± 22.50, 9.36 ± 5.50 and 12.17 ± 13.44 resectively. It was also found that the level of DNMT3b mRNA in CML significantly increased in comparison with NC (P < 0.05), and that the between the 3 different phase of CML was not statistically significantly different (P >0.05). The expression of DNMT mRNA increases in advanced CML as compared with normal controls and CML-CP, and the increased levels of DNMT mRNA probably correlate with disease progression in CML.

Coordination of AUF1 and miR-148a destabilizes DNA methyltransferase 1 mRNA under hypoxia in endometriosis.

DNA methylation is regulated by hypoxia in endometriosis. Hypoxia causes global hypomethylation through AU-rich element binding factor 1 (AUF1)/microRNA-148a (miR-148a)-mediated destabilization of DNA methyltransferase 1 (DNMT1) mRNA. Eutopic endometrial and ectopic endometriotic stromal cells have the same genetic background, but differ in several cellular and molecular responses. Both hypoxia and DNA methylation regulate several genes involved in the development of endometriosis. This laboratory study included 15 patients of reproductive age with endometriosis or normal menstrual cycles. Paired endometrial and endometriotic tissues were collected for assaying the levels of DNMT1, 3a and 3b using quantitative RT-PCR, western blot and immunohistochemical (IHC) staining. Primary cultured endometrial stromal cells maintained in normoxia/hypoxia (1% O2) or treated with hypoxia-mimetic compounds were also assayed. The levels of DNA 5-methylcytosine were assayed by using IHC in clinical specimens and murine tissues, and by ELISA in cultured stromal cells. The 3'-untranslated region reporter assay was used to evaluate the effect of hypoxia, microRNAs (miRNAs) and human antigen R (HuR)/AUF1 on DNMT1 mRNA stability. RNA immunoprecipitation was used to assess the interaction of HuR/AUF1 and miR-148a/DNMT1 mRNA under hypoxia. Finally, a transplant-induced mouse model of endometriosis using 20 mice was used to elucidate the alteration of Dnmt1 levels and DNA methylation in the endometriotic tissues. Levels of DNMT1 mRNA and protein and 5-methylcytosine were lower in the ectopic stromal cells (P < 0.05) than in the eutopic cells. Treatment with hypoxia and its mimetic compounds recapitulated the reduced levels of DNMT1 and 5-methylcytosine levels (P < 0.05 versus control). Hypoxia treatment destabilized DNMT1 mRNA through recruitment of miR-148a and AUF1. Mutations introduced to the miR-148a targeting site or AU-rich element (ARE) restored the hypoxia-suppressed DNMT1 3'-untranslated region (3'-UTR) reporter activity (P < 0.05 versus control). Levels of proteins of three hypermethylated genes in endometrial stroma cells, GATA6, HOXA3 and SLC16A5, were elevated after 72 h of hypoxia treatment (P < 0.05 versus control). Finally, a transplant-induced model of endometriosis demonstrated the down-regulation of DNMT1 and a decrease in 5-methylcytosine in the endometriotic tissues (P < 0.05, eutopic versus ectopic). Primary human cell cultures and a murine model were used in this study, and thus the results may not fully represent the situation in vivo. This is the first study to elucidate how microenvironmental hypoxia links to the epigenetic effects of DNA methylation in the endometriosis, and to delineate the molecular mechanism of hypoxia-coordinated AUF1/miR-148a interaction and recruitment to DNMT1 mRNA during the pathogenesis of endometriosis. The development of future therapeutics in endometriosis may aim at disrupting this specific interaction and eventually restore the epigenetic regulation. This work was supported by the National Science Council of Taiwan (NSC101-2320-B-006-030-MY3). The author declares that there are no conflicts of interest.

E6 and E7 gene silencing results in decreased methylation of tumor suppressor genes and induces phenotype transformation of human cervical carcinoma cell lines.

In SiHa and CaSki cells, E6 and E7-targeting shRNA specifically and effectively knocked down human papillomavirus (HPV) 16 E6 and E7 at the transcriptional level, reduced the E6 and E7 mRNA levels by more than 80% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in down-regulation of DNA methyltransferase mRNA and protein expression, decreased DNA methylation and increased mRNA expression levels of tumor suppressor genes, induced a certain apoptosis and inhibited proliferation in E6 and E7 shRNA-infected SiHa and CaSki cells compared with the uninfected cells. Repression of E6 and E7 oncogenes resulted in restoration of DNA methyltransferase suppressor pathways and induced apoptosis in HPV16-positive cervical carcinoma cell lines. Our findings suggest that the potential carcinogenic mechanism of HPV16 through influencing DNA methylation pathway to activate the development of cervical cancer exist, and maybe as a candidate therapeutic strategy for cervical and other HPV-associated cancers.

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.

Down-regulation of hsa-miR-1264 contributes to DNMT1-mediated silencing of SOCS3.

Previously we found decreased expression of SOCS3 in neointimal hyperplastic region following balloon angioplasty in atherosclerotic micro swine. In our recent in vitro studies using human coronary artery smooth muscle cells (HCASMC), we observed the inhibition of SOCS3 expression in the presence of both TNF-α and IGF-1, correlating with the in vivo findings in microswine. We also reported that two independent mechanisms, JAK/STAT3/NFκB and promoter methylation of SOCS3 were responsible for TNF-α and IGF-1 induced SOCS3 inhibition. In this study, using miRNA array and gene expression approaches, we explored the molecular mechanisms involved in the above SOCS3 repression and identified several miRNAs that are associated with the regulation of SOCS3 expression. Our miRNA expression profiling revealed profound down-regulation of two specific miRNAs, hsa-miR-758 and hsa-miR-1264, whose expression levelswere decreased by 8-10 folds inHCASMCs thatwere treated with both TNF-α and IGF-1. This was accompanied with a significant up-regulation of three specific miRNAs, hsa-miR-155, hsa-miR-146b-5p and hsa-miR-146a, which showed about 3-7 fold increases in their expression levels. Importantly, we also found that the miRNA hsa-miR-1264 targets DNA methyltransferase-1 (DNMT1) transcripts by binding to its 3'UTR region to affect its expression. Expression of hsa-miR-1264 in HCASMCs not only resulted in decreased DNMT1 mRNA transcripts but it also increased SOCS3 expression. The treatment with TNF-α and IGF-1 resulted in drastic decrease in hsa-miR-1264 levels with no change in the expression of DNMT1. Consequently, the DNMT1 activity caused hypermethylation in the CpG island of the SOCS3 promoter region and inhibited its expression. This could be a causative epigenetic mechanism associated with TNF-α and IGF-1 induced smooth muscle cell proliferation involved in the pathogenesis of coronary artery hyperplasia and restenosis.

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

Downregulation of DNA methyltransferase 1 in zona-free cloned buffalo (Bubalus bubalis) embryos by small interefering RNA improves in vitro development but does not alter DNA methylation level.

Aberrant epigenetic reprogramming, especially genomic hypermethylation, is implicated as the primary reason behind the failure of the cloning process during somatic cell nuclear transfer (SCNT). We transfected one-cell-stage zona-free buffalo embryos produced by handmade cloning with 50 nM DNMT1 small interfering RNA (siRNA), using lipofectamine, to knockdown the DNA methyltransferase 1 (DNMT1) gene. siRNA treatment decreased (p<0.001) the expression level of DNMT1 mRNA and DNMT1 protein in the one-cell-stage embryos and increased (p<0.05) the blastocyst rate (52.3 ± 1.3% vs. 45.3 ± 2.5%) compared to that in the controls, but did not reduce the DNA methylation level similar to the in vitro-fertilized (IVF) embryos. It also increased (p<0.05) the relative mRNA abundance of P53 and CASPASE 3, but not that of HDAC1, DNMT1, and DNMT3a, in the blastocysts of the siRNA group compared to the controls. The global level of H3K18ac was higher (p<0.05) in the blastocysts of the siRNA group than in the controls, whereas that of H3K9ac and H3K27me3 was not significantly different between the two groups. In conclusion, lipofection can be successfully used for transfection of DNMT1 siRNA into one-cell-stage zona-free cloned buffalo embryos. It results in a concomitant decrease in the DNMT1 mRNA and protein levels in the one-cell-stage embryos. siRNA-mediated knockdown increases the blastocyst rate but does not alter the DNA methylation level.

Influence of gossypol acetic acid on the growth of human adenoid cystic carcinoma ACC-M cells and the expression of DNA methyltransferase 1.

We investigated the effects of gossypol acetic acid (GAA) on the proliferation, apoptosis, and expression of DNA methyltransferase 1 (DNMT1) mRNA in human adenoid cystic carcinoma (ACC-M) cells in vitro. The proliferation and apoptosis of ACC-M cells after treatment with different concentrations of GAA were detected using Cell Counting Kit-8 and flow cytometry, respectively. DNMT1 mRNA expression was measured by real-time fluorescence quantitative polymerase chain reaction. The growth of ACC-M cells was inhibited after treatment with GAA for 24, 48, and 72 h. The apoptotic rates of ACC-M cells after treatment with GAA for 72 h were higher than those of control cells (without treatment) (P < 0.05). DNMT1 mRNA expression in ACC-M after treatment with GAA for 72 h was lower than that in control cells (P < 0.05). GAA had inhibitory effects on the proliferation and induced apoptosis of human ACC-M cells, while GAA also reduced the expression level of DNMT1 mRNA in ACC-M cells.

Effects of atrazine and chlorpyrifos on DNA methylation in the brain and gonad of the common carp

DNA methylation is known to play an important role in the regulation of gene expression in animal. The purpose of the present study was to examine the effect of atrazine (ATR), chlorpyrifos (CPF) and combined ATR/CPF exposure on DNA methylation in the brain and gonad of common carp (Cyprinus carpio L.). The carp were sampled after a 40-d exposure to CPF and ATR, individually or in combination, followed by a 40-d recovery to measure the levels of global DNA methylation and the expression of methylation enzymes (DNA methyltransferases (DNMTs) and methylcytosine binding domain 2 (MBD2)) in the brain and gonad tissues. The results revealed that a significant global DNA hypomethylation in the common carp exposed to ATR, CPF and their mixture was observed compared to the control fish. The MBD2 mRNA expression was up-regulated in the brain and gonad of the common carp exposed to ATR, CPF and their mixture, in contrast, the DNMTs mRNA expression was down-regulated. The information regarding the effects of ATR and CPF on DNA methylation status generated in this study is important for pesticides toxicology evaluation. However, the effect of ATR and CPF on the methylation status of specific genes, as well as its detailed mechanism requires further investigation.

Effects of acute ethanol exposure on anxiety measures and epigenetic modifiers in the extended amygdala of adolescent rats.

Epigenetic mechanisms appear to play an important role in neurodevelopment. We investigated the effects of acute ethanol exposure on anxiety measures and function of histone deacetylases (HDAC) and DNA methyltransferases (DNMT) in the amygdala and bed nucleus of stria terminalis (BNST) of adolescent rats. One hour after ethanol exposure, rats were subjected to anxiety measures. A subset of adolescent rats was exposed to two doses (24 h apart) of ethanol (2 g/kg) to measure rapid ethanol tolerance to anxiolysis. The HDAC and DNMT activities and mRNA levels of DNMT isoforms were measured in the amygdala and BNST. The lower dose of ethanol (1 g/kg) produced neither anxiolysis, nor inhibited the HDAC and DNMT activities in the amygdala and BNST, except DNMT activity in BNST was attenuated. Anxiolysis by ethanol was observed at 2 and 2.25 g/kg, whereas higher doses (2.5 and 3 g/kg) were found to be sedative. DNMT activity in the amygdala and BNST, and nuclear HDAC activity in the amygdala, but not in the BNST were also inhibited by these doses of ethanol. A lack of tolerance was observed on ethanol-induced inhibition of DNMT activity in the amygdala and BNST, and nuclear HDAC activity in the amygdala, as well to anxiolysis produced by ethanol (2 g/kg). The DNMT1, DNMT3a, and DNMT3b mRNA expression in the amygdala was not affected by either 1or 2 doses of 2 g/kg. However, DNMT1 and DNMT3a expression in the BNST was increased, whereas DNMT3l mRNA was decreased in the amygdala, after 2 doses of 2 g/kg ethanol. These results suggest that reduced sensitivity to anxiolysis and the lack of rapid tolerance to the anxiolytic effects of ethanol and inhibition of HDAC and DNMT functions may play a role in engaging adolescents in binge drinking patterns.

Global DNA hypomethylation: A potential mechanism in King pigeon nerve tissue damage induced by avermectin

As an effective insecticidal and nematicidal agent, avermectin (AVM) has been widely used in agricultural production and stock farming areas. Subsequently, the residues of AVM or its active metabolites in animal manure pose a toxic threat to non-target organisms in the environment. As the most characteristic epigenetic phenomena, DNA methylation status is a useful biological signal for the toxicity assessment of environmental chemical toxicants. In this study, analyses of the overall level of genomic DNA methylation were performed, and the expression levels of DNA methyltransferases (DNMTs), as well as demethylase methyl-CpG-binding domain protein 2 (MBD2), in pigeon brain tissues after subchronic exposure (with a AVM concentration of 20mg/kg, 40mg/kg and 60mg/kg, respectively) to AVM for 30, 60 and 90days were investigated. Global DNA hypomethylation and down-regulation of DNMT mRNA expression occurred in a dose–time-dependent manner in pigeon brains. The expression level of MBD2, which functions as a demethylase, was significantly enhanced in a dose-dependent but not time-dependent manner. In addition, the elevated expression level of MBD2 had a more robust effect on genomic DNA hypomethylation compared to changes in DNMT expression. Taken together, these results suggested that subchronic dose exposures of AVM could affect the global DNA methylation status, and this mechanism is closely related to changes in the expression levels of DNMTs and MBD2.

Folate Deficiency and Aberrant Expression of DNA Methyltransferase 1 were Associated with Cervical Cancerization

DNA methyltransferase 1 (DNMT1) plays a significant role in maintaining DNA methylation. Aberrant DNA methylation is a recognized feature of human cancers and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also have suggested that folate deficiency was associated with many cancers. The aim of the present study was to evaluate the effect of folate deficiency and aberrant expression of DNA methyltransferase 1 (DNMT1) on cervical cancerization. The expression of DNMT1 protein and mRNA and levels of serum folate were detected in 238 women with a diagnosis of normal cervix (NC，n = 53), cervical intraepithelial neoplasia (CIN I, n = 52; CIN II/III, n = 53), and squamous cell carcinoma of the cervix (SCC; n = 80). In addition, the expression of DNMT1 protein and mRNA was measured in cervical cancer cells (Caski and C33A) treated by different concentration of folate. Serum folate levels decreased and expression levels of DNMT1 protein and mRNA increased gradually with progressive severity of the cervix lesions (P<0.001). It was found that folate was able to reduce the viability of Caski or C33A cell (r=0.978, P=0.002; r=0.984, P<0.001) and regulated aberrant expression of DNMT1 protein (r=-0.859, P=0.01; r=-0.914, P<0.001) and mRNA (r=-0.297, P=0.159; r=0.433, P=0.034) in vitro. Our findings indicated that the low-level of serum folate and high-expression of DNMT1 protein or mRNA was significantly associated with cervical carcinogenesis. Folate deficiency and aberrant expression of DNA methyltransferase 1 had additive effect on cervical cancerization. Folate supplement and recovery of aberrant DNA methylation status may offer a new strategy for prevention and therapy of cancers.

MicroRNA-148b and microRNA-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines

Overexpression of DNA methyltransferase 1 (DNMT-1) is observed mostly in pancreatic cancer and it can cause tumor suppressor genes silencing in this disease. Recent studies suggest that abnormal expressions of microRNAs (miRs) are involved in pathogenesis of different types of human cancers including pancreatic cancer. In this study we aimed to investigate the effect of miR-148b and -152 on reverting the tumorigenic phenotype of pancreatic cancer cell lines. In order to investigate whether miR-148b and -152 are involved in the regulation of DNMT-1, luciferase reporter assay was used and confirmed that the DNMT-1 mRNA could be a target for miR-148b and miR-152. Furthermore, overexpression of miR-148b and -152 in pancreatic cancer cell lines (MIA PaCa-2 and AsPC-1) decreased DNMT-1 expression (53% and 59% respectively), returned DNA methylation to normal patterns and induced re-expression of tumor suppressor genes, like BNIP3 (4.7- and 3.8-fold) and SPARC (5.3- and 2.9-fold) for miR-148b and -152 respectively. Moreover, the introduced miR-148b and -152 could inhibit the proliferation of MIA PaCa-2 (35% and 37% respectively) and AsPC-1 (39% and 40% respectively) cell lines. The apoptosis rates of MIA PaCa-1 after treatment with miR-148b and -152 were 10% and 8% respectively; while these rates in AsPC-1 were 16% and 11% respectively. Conclusively these findings mean that miRs that are targeting DNMT-1 and modifying methylation status of tumor suppressor genes such as BNIP3 and SPARC can be applied in killing the pancreatic cancer cells and decreasing the tumorigenicity of these cells.

Effect of DNA methyltransferase 1 and methyl-CpG-binding protein 2 abnormal expression on cervical lesions and related interaction

To explore the effect of DNA methyltransferase 1 (DNMT1) and methyl-CpG-binding protein 2 (MeCP2) on cervical cancer and cervix precancerous lesion. 74 patients with cervix squamous cell carcinoma(SCC), 52 patients with cervical intraepithelial neoplasm I (CIN I), 60 patients with cervical intraepithelial neoplasm II - II (CIN II-III)and 58 patients with histologically diagnosed cervix inflammation(CI), were included in this study. Information as demography, reproductive history, life style, HPV infection were collected. Western Blot were used to detect the expression of DNMT1 protein and MeCP2 protein. Real-time PCR was used to detect the expression of DNMT1 and MeCP2 mRNA. Levels of DNMT1 and MeCP2 protein expression increased gradually with the deterioration of cervical lesion (H = 94.33, P < 0.001;F = 21.580, P < 0.001). Along with the deterioration of cervical lesion, levels of DNMT1 and MeCP2 mRNA expression were gradually increasing( F = 4.758, P = 0.003; F = 7.804, P < 0.001). Data from Correlation analysis showed that both protein (r = 0.287, P < 0.001) and mRNA(r = 0.179, P = 0.005)were positive correlated with DNMT1 and MeCP2. of our study indicated that there was an additive interaction between high-expression of DNMT1 protein and high-expression of MeCP2 protein in SCC or CIN II-III. However, there was an additive interaction between high-expression of DNMT1 mRNA and high-expression of MeCP2 mRNA in SCC or CIN II-III. Results from our study revealed the fact that both high expression of DNMT1 protein and high expression of MeCP2 protein could increase the risk of cervix cancerization. According to our findings, there might be a synergistic action existed between DNMT1 and MeCP2 during the progression of cervix cancelation.

Role of NADPH oxidase NOX5-S, NF-κB, and DNMT1 in acid-induced p16 hypermethylation in Barrett's cells

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.

DNA methytransferases are differentially expressed in human eyes

AbstractPurpose We studied DNA methyltransferases (DNMT) implicated in CpG methylation pattern. The aim of our work was to extensively check the presence of DNMT (mRNA and protein levels) and quantify their expressions in the anterior segment.Methods mRNAs were extracted from human cornea, conjunctiva, capsule, trabeculum and from related cell lines (HCE, HTCE, CHANG and TM). cDNAs were generated by reverse transcription, quantified by amplification with specific primers for DNMT1, 3A, 3B and 3L and normalized to the housekeeping gene 36B4. For immunohistochemistry, normal human corneas were provided by the regional corneal graft bank. All proteins were detected by specific primary antibody for DNMT1, 3A, 3B and 3L (Santa‐Cruz®), negative control were done with a rabbit IgG antibody as primary one. Secondary antibody Cy3‐coupled was used.Results All DNMT are expressed in the human eyes but at different levels. DNMT3L looks like to be the weakly expressed. All tested cell lines also expressed DNMTs. Quantitative RT‐PCR demonstrated strong differences between members and zones of expression of these DNMTs. Our transcripts results were confirmed by immunohistochemistry. All tested cell lines also expressed DNMTs. Quantitative RT‐PCR demonstrated strong differences between members and zones of expression of these DNA methyltransferases. Our transcripts results were confirmed by immunohistochemistry experiments on cornea sections.Conclusion For the first time, an inventory of the DNMT expression in tissues and cells lines from human ocular anterior segment was performed. Future studies will certainly permit to demonstrate the presence of a direct link between a deregulation of the CpG methylation pattern and the already “well known” eye pathologies.

Ultraviolet B enhances DNA hypomethylation of CD4+ T cells in systemic lupus erythematosus via inhibiting DNMT1 catalytic activity

BackgroundCD4+ T cells DNA hypomethylation is involved in the pathogenesis of systemic lupus erythematosus (SLE). Recent studies showed that ultraviolet B (UVB, 290–320nm) might induce the exacerbation of SLE by decreasing the DNA methylation level. However, the role of DNA methyltransferase 1 (DNMT1) in the UVB-induced CD4+ T cells DNA hypomethylation remains unclear. ObjectiveTo elucidate the role of DNMT1 in lupus CD4+ T cells global DNA hypomethylation enhanced by UVB. Methods35 SLE patients and 15 healthy controls were enrolled in the study. CD4+ T cells from SLE patients and healthy controls exposed to different dosages of UVB were analyzed. The global DNA methylation measurement, real-time PCR, Western blotting and DNMT1 catalytic activity detection were employed. ResultsThe level of global DNA methylation and DNMT1 mRNA expression in CD4+ T cells from SLE patients were significantly lower than those from the control group. DNA methylation was decreased after UVB exposure in a dosage-dependent manner in SLE patients, but not in the control group. DNMT1 mRNA and protein expression level were not affected by UVB exposure in both SLE patients and healthy controls. DNMT1 catalytic activity was significantly decreased in CD4+ T cells from SLE patients after UVB exposure in a dosage-dependent manner. DNMT1 catalytic activity was lower and more sensitive to UVB exposure in CD4+ T cells from active SLE patients that from stable ones. ConclusionUVB enhanced DNA hypomethylation of CD4+ T cells in SLE via inhibiting DNMT1 catalytic activity in a dosage-dependent manner.

Expression and significance of DNMT1 in human keloid fibroblast

To investigate the expression and significance of DNA methyltransferase 1 (DNMT1) in different parts and formation time of keloid fibroblasts. Specimen were collected from fifty-three patients with keloid. The keloids were grouped according to formation time. Immunohistochemistry was used to assay the expression of DNMT1 in normal skin, invasive part, proliferative part and aged part. RT-PCR was adopted to assay the expression of DNMT1 mRNA in each group. DNMT1 is mainly distributed in the nucleus of fibroblasts. The expression of DNMT1 protein and DNMTl mRNA in keloid (72%) is higher than that in normal skin fibroblasts (8%) (P = 0.001). The expression rate of DNMT1 protein and DNMT1 mRNA in shorter formation time group (100%, Score:10.47 +/- 1.85) is higher than a longer formation time group (45%,Score: 7.71 +/- 1.80) (P = 0.007). The expression rate of DNMT1 protein and DNMT1 mRNA in the invasive part of keloid (100%, Score: 10.47 +/- 1.85) is higher than the proliferative part (78%, Score: 6.63 +/- 1.75) and aged part (38%, Score: 5.53 +/- 1.55) (P = 0.001). DNMT1 plays an important role in keloid. The expression of DNMT1 tends to decrease along with the increase of the formation time in keloid. It is also related to the part of keloid. DNMT1 plays an important role in the process of keloid growth and is closely related to infiltrative growth in keloid.

Effects of ultraviolet B exposure on DNA methylation in patients with systemic lupus erythematosus

The aim of this study was to investigate the effects of ultraviolet B (UVB) exposure on DNA methylation in patients with systemic lupus erythematosus (SLE) and its significance in the pathogenesis of SLE. T cells from 35 SLE patients and 21 healthy individuals were cultured and irradiated with UVB. The global DNA methylation profiles of the T cells obtained from the patients and controls following irradiation with UVB were assessed using specific monoclonal antibodies for 5-methylcytosine and analyzed quantitatively through flow cytometry. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the levels of DNA methyltransferase 1 (DNMT1) and methyl CpG binding domain protein 2 (MBD2) in T cells from the patients and controls following UVB irradiation. Significant global DNA hypomethylation was observed in the SLE patients compared with the controls (P<0.01). The SLE patients also had significantly lower levels of DNMT1 mRNA expression (P<0.01) and significantly higher levels of MBD2 mRNA compared with the controls (P<0.01). DNA methylation was decreased following UVB irradiation at two different dosages and the DNA methylation levels of the patients with active SLE were more sensitive to UVB. The level of DNMT1 mRNA was decreased following UVB irradiation at the higher dosage in the patients with active SLE, but no significant difference was observed in MBD2 mRNA expression. UVB exposure is able to inhibit DNA methylation and DNMT1 mRNA expression, which is subsequently involved in the epigenetic mechanism of SLE. The process by which DNA hypomethylation occurs in patients with SLE is complicated and the multiple factors that are involved in DNA methylation and demethylation events require further study.