methyl-CpG-binding Domain Protein 2 Expression Research Articles

MBD2 mediates Th17 cell differentiation by regulating MINK1 in Th17-dominant asthma.

Objectives: .Asthma is a highly heterogeneous disease, and T-helper cell type 17 (Th17) cells play a pathogenic role in the development of non-T2 severe asthma. Misshapen like kinase 1 (MINK1) is involved in the regulation of Th17 cell differentiation, but its effect on severe asthma remains unclear. Our previous studies showed that methyl-CpG binding domain protein 2 (MBD2) expression was significantly increased in patients with Th17 severe asthma and could regulate Th17 cell differentiation. The aim of this study was to investigate how MBD2 interacts with MINK1 to regulate Th17 cell differentiation in Th17-dominant asthma. Materials and methods: Female C57BL/6 mice and bronchial epithelial cells (BECs) were used to establish mouse and cell models of Th17-dominant asthma, respectively. Flow cytometry was used to detect Th17 cell differentiation, and the level of IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). Western blot and quantitative real-time PCR (qRT-PCR) were used to detect MBD2 and MINK1 expression. To investigate the role of MBD2 and MINK1 in Th17 cell differentiation in Th17-dominant asthma, the MBD2 and MINK1 genes were silenced or overexpressed by small interfering RNA and plasmid transfection. Results: Mouse and BEC models of Th17-dominant asthma were established successfully. The main manifestations were increased neutrophils in BALF, airway hyperresponsiveness (AHR), activated Th17 cell differentiation, and high IL-17 levels. The expression of MBD2 in lung tissues and BECs from the Th17-dominant asthma group was significantly increased, while the corresponding expression of MINK1 was significantly impaired. Through overexpression or silencing of MBD2 and MINK1 genes, we have concluded that MBD2 and MINK1 regulate Th17 cell differentiation and IL-17 release. Interestingly, MBD2 was also found to negatively regulate the expression of MINK1. Conclusion: Our findings have revealed new roles for MBD2 and MINK1, and provide new insights into epigenetic regulation of Th17-dominant asthma, which is dominated by neutrophils and Th17 cells. This study could lead to new therapeutic targets for patients with Th17-dominant asthma.

Androgen Plays a Potential Novel Hormonal Therapeutic Role in Th17 Cells Predominant Neutrophilic Severe Asthma by Attenuating BECs Regulated Th17 Cells Differentiation via MBD2 Expression.

T helper 17 (Th17) cells subtype of non-T2 asthma is less responsive (resistant) to inhaled corticosteroids (ICS), so also called severe asthma. Methyl-CpG-binding domain protein 2 (MBD2) regulates the differentiation of the Th17 cells, showing the possibility of a therapeutic target in severe asthma. Androgen tends to show beneficial therapeutic effects and is a "hot research topic," but its role in the differentiation and expression of Th17 cells via MBD2 is still unknown. The aim of this study was to evaluate how sex hormone interacts with MBD2 and affects the differentiation and expression of Th17 cells in severe asthma. Here, first, we measured the concentration of androgen, estrogen, and androgen estrogen ratio from subjects and correlated it with severe asthma status. Then, we established an animal model and bronchial epithelial cells (BECs) model of severe asthma to evaluate the role of MBD2 in the differentiation and expression of Th17 cells (IL-17), the therapeutic potential of sex hormones in severe asthma, and the effect of sex hormones in BECs regulated Th17 cells differentiation via MBD2 at the cellular level. Increased MBD2 expression and Th17 cells differentiation were noted in the animal and the BECs severe asthma models. Th17 cell differentiation and expression were MBD2 dependent. Androgen attenuated the differentiation of BECs regulated Th17 cells via MBD2 showing BECs as a therapeutic target of androgen, and these findings postulate the novel role of androgen in Th17 cells predominant neutrophilic severe asthma therapy through targeting MBD2.

Increased Methyl-CpG-Binding Domain Protein 2 Promotes Cigarette Smoke-Induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a chronic vascular proliferative disorder. While cigarette smoke (CS) plays a vital part in PH related to chronic obstructive pulmonary disease (COPD). Methyl-CpG-Binding Domain Protein 2 (MBD2) has been linked to multiple proliferative diseases. However, the specific mechanisms of MBD2 in CS-induced PH remain to be elucidated. Herein, the differential expression of MBD2 was tested between the controls and the PH patients’ pulmonary arteries, CS-exposed rat models’ pulmonary arteries, and primary human pulmonary artery smooth muscle cells (HPASMCs) following cigarette smoke extract (CSE) stimulation. As a result, PH patients and CS-induced rats and HPASMCs showed an increase in MBD2 protein expression compared with the controls. Then, MBD2 silencing was used to investigate the function of MBD2 on CSE-induced HPASMCs’ proliferation, migration, and cell cycle progression. As a consequence, CSE could induce HPASMCs’ increased proliferation and migration, and cell cycle transition, which were suppressed by MBD2 interference. Furthermore, RNA-seq, ChIP-qPCR, and MassARRAY were conducted to find out the downstream mechanisms of MBD2 for CS-induced pulmonary vascular remodeling. Subsequently, RNA-seq revealed MBD2 might affect the transcription of BMP2 gene, which furtherly altered the expression of BMP2 protein. ChIP-qPCR demonstrated MBD2 could bind BMP2’s promotor. MassARRAY indicated that MBD2 itself could not directly affect DNA methylation. In sum, our results indicate that increased MBD2 expression promotes CS-induced pulmonary vascular remodeling. The fundamental mechanisms may be that MBD2 can bind BMP2’s promoter and downregulate its expression. Thus, MBD2 may promote the occurrence of the CS-induced PH.

Loss of MBD2 ameliorates LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis.

Apoptosis of alveolar epithelial cells is a critical initial link in the pathogenesis of acute lung injury (ALI), recent studies have revealed that Methyl-CpG binding domain protein 2 (MBD2) was involved in the execution of apoptosis, yet its role in ALI remained unclear. In the present study, we aim to explore the role and mechanism of MBD2 in the pathogenesis of ALI. We have found that MBD2 expression, in parallel to apoptosis, increased in alveolar epithelial cells of mice treated with LPS, knockout of MBD2 reduced apoptosis and protected mice from LPS-induced ALI. In MLE-12 cells, a cell line of murine alveolar epithelial cells, LPS induced MBD2 expression and apoptosis in a dose- and time-dependent manner. Knockdown of MBD2 with shRNA alleviated, while overexpression of MBD2 increased LPS-induced apoptosis. Mechanistically, intracellular zinc level decreased when MLE-12 cells were treated with LPS. MBD2knockdown restored intracellular zinc level after LPS treatment, and MBD2 overexpression further aggravated LPS-induced intracellular zinc loss. Metal transcription factor 1 (MTF1) is a critical transcription factor in charge of intracellular zinc efflux. LPS treatment induced MTF1 expression both in vivo and in vitro. Inhibition of MTF1 reduced LPS-induced apoptosis in MLE-12 cells. MBD2 could bind to the promoter region of MTF1 and promote MTF1 expression. Collectively, these data indicated that loss of MBD2-ameliorated LPS-induced alveolar epithelial cell apoptosis and ALI in mice via modulating intracellular zinc homeostasis by upregulating MTF1.

Silencing of MBD2 and EZH2 inhibits the proliferation of colorectal carcinoma cells by rescuing the expression of SFRP.

The secreted frizzled related proteins (SFRPs) are extracellular inhibitors of WNT pathway signaling. Methyl-CpG binding domain protein 2 (MBD2) and enhancer of zeste homolog 2 (EZH2) are core members of the methylated DNA binding domain (MBD) and polycomb group (PcG) protein families for epigenetic regulation, respectively. This study aimed to ascertain the potential role of MBD2 and EZH2 proteins in colorectal cancer (CRC) and its effects on the expression of SFRP. Bioinformatics, real-time quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect the expression of MBD2, EZH2, and SFRP in CRC cell lines and tissues. The functions of MBD2 and EZH2 in regards to cell proliferation, cell cycle distribution, apoptosis and invasion were examined in CRC cell lines. Methylation-specific PCR (MSP) was used to detect the methylation status of the SFRP promoter. The results revealed that the mRNA expression levels of SFRP were significantly decreased in CRC tissues and cell lines compared to these levels in the adjacent tissues and NCM460, respectively. However, the mRNA levels of EZH2 and MBD2 genes were highly expressed in CRC cell lines. We found that reducing MBD2 and EZH2 expression together remarkably inhibited and decreased the proliferation, migration and invasion abilities of the CRC cell lines compared to reducing one of each. Flow cytometric analysis showed that knockdown of MBD2 and EZH2 together in CRC affected cell apoptosis and the cell cycle progression more effectively than knockdown of one of each. The mRNA expression of SFRP1 was reactivated by silencing of MBD2 but not EZH2 in SW480 and HCT116 cells. SFRP4 and SFRP5 mRNA expression was reactivated by silencing of EZH2 but not MBD2 only in SW480 cells. However, depletion of both MBD2 and EZH2 restored SFRP1, SFRP2, SFRP4, and SFRP5 mRNA expression more effectively in CRC cells. Interestingly, there was no significant change in the methylation status of SFRP1, SFRP2, SFRP4, and SFRP5 gene promoter between before and after interference with MBD2, EZH2, and both. In conclusion, our results suggest that silencing of MBD2 and EZH2 simultaneously was able to rescue the expression of SFRP and inhibit the proliferation of CRC cells more effectively. However, the underlying regulatory mechanism system of MBD2 and EZH2 for SFRP in CRC requires further research.

MBD2 as a Potential Novel Biomarker for Identifying Severe Asthma With Different Endotypes.

Background: Studies have shown that methyl-CpG binding domain protein 2 (MBD2) expression is significantly elevated in a neutrophil-dominant severe asthma mouse model. It also regulates Th17 cell differentiation. The objective of this study was to investigate the relationship between serum MBD2 levels in patients with severe asthma with different endotypes.Methods: Eligible adults with confirmed asthma (n = 63) underwent a clinical assessment, asthma control test and pulmonary function test and were classified as having mild, moderate or severe asthma. Severe asthma endotypes were defined according to the percentage of Th2 and Th17 cells in the peripheral blood and by the type of inflammation. The percentage of Th2 and Th17 cells in the peripheral blood was determined by flow cytometry. Serum MBD2, eosinophilic cationic protein and myeloperoxidase were measured by enzyme-linked immunosorbent assay. Correlations of MBD2 expression with clinical parameters were evaluated using Spearman's rank correlation analysis.Results: Serum MBD2 levels were upregulated in patients with severe asthma compared to healthy controls and patients with mild to moderate asthma. MBD2 was also significantly increased in patients with Th17 severe asthma compared to patients with type 2 severe asthma. Furthermore, MBD2 was positively correlated with MPO and Th17 cells but negatively correlated with ECP and Th2 cells in patients with severe asthma.Conclusions: These findings suggest that serum MBD2 may be a potential new biomarker for identifying severe asthma, Th17 severe asthma and the type of airway inflammation. However, these findings are still preliminary and need to be further investigated.

Mechanisms by Which the MBD2/miR-301a-5p/CXCL12/CXCR4 Pathway Regulates Acute Exacerbations of Chronic Obstructive Pulmonary Disease.

BackgroundChronic obstructive pulmonary disease (COPD) is characterized by irreversible expiratory airflow obstruction, and its chronic course is worsened by recurrent acute exacerbations. Our previous microarray assay identified microRNA (miR)-301a-5p as being associated with progression of acute exacerbation of COPD (AE-COPD); however, the mechanism underlying COPD pathogenesis remains unknown.MethodsSamples of serum and peripheral blood mononuclear cells (PBMCs) were isolated from healthy control subjects and patients with stable COPD (R-COPD) or with an acute exacerbation of COPD (AE-COPD). Human HULEC-5a and human bronchial epithelial (HBE) cells were transfected with methyl-CpG-binding domain protein 2 (MBD2), sh-MBD2, miR-301a-5p mimics or an inhibitor, and then stimulated with cigarette smoke extract (CSE). Conditioned medium co-culture assays were performed by adding the supernatant of medium derived from HULEC-5a cells transfected with miR-301a-5p mimics or inhibitor into wells containing si-c-x-c motif chemokine receptor 4 (CXCR4)-transfected-lung fibroblasts or human leukemic THP-1 cell line macrophages. Transwell assays were performed to analyze cell migration.ResultsOur analysis of clinical samples showed that decreased miR-301a-5p levels in patients with AE-COPD were positively correlated with levels of MBD2 expression, but negatively correlated with levels of chemokine ligand C-X-C motif chemokine ligand 12 (CXCL12) expression. MBD2 overexpression significantly promoted miR-301a-5p production, but suppressed CXCL12 production in HULEC-5a and HBE cells. CXCL12 was confirmed to be a direct target of miR-301a-5p. CXCR4 knockdown significantly enhanced the suppressive effect of miR-301a-5p mimics and attenuated the promotional effects of the miR-301a-5p inhibitor on the migration of circulating fibroblasts and macrophages, as well as the expression levels of phospho-mitogen-activated protein kinase (p-MEK) and phospho-protein kinase B (p-AKT).ConclusionIn summary, the MBD2/miR-301a-5p/CXCL12/CXCR4 pathway was shown to affect the migration of lung fibroblasts and monocyte-derived macrophages, which may play an important role during COPD exacerbations.

Expression and clinical significance of methyl-CpG binding domain protein 2 in high-grade serous ovarian cancer.

Platinum resistance is an important cause of clinical recurrence and mortality of patients with high-grade serous ovarian cancer (HGSOC). Methyl-CpG binding domain protein 2 (MBD2) serves an important role in tumor progression; however, its role in HGSOC remains unclear. The aim of the present study was to investigate the expression of MBD2 in HGSOC and its role in drug resistance and prognosis of HGSOC. MBD2 expression was analyzed by immunohistochemical staining and western blotting. The associations between MBD2 expression and clinical pathological features, platinum resistance and patient prognosis were analyzed using a χ2 test, Kaplan-Meier analysis and Cox regression analysis. Positive MBD2 expression was detected in 73 (63.5%) of the HGSOC tissue samples, whereas it was undetectable in all 16 normal tissue samples (100%) analyzed, indicating a significantly higher expression level in tumor tissues compared with normal tissues (P<0.001). Additionally, MBD2 expression was significantly higher in platinum-resistant cases compared with that in platinum-sensitive cases (P<0.05). In addition, high expression of MBD2 was negatively associated with relapse-free survival (P<0.05). In conclusion, MBD2 was demonstrated to be a potential drug target and a biomarker for poor prognosis in HGSOC.

Reduced MBD2 expression enhances airway inflammation in bronchial epithelium in COPD.

BackgroundChronic obstructive pulmonary disease (COPD) is a common inflammatory lung disease characterized by inflammatory cells activation and production of inflammatory mediators. Methyl-CpG-binding domain protein 2 (MBD2) plays an important role in diverse immunological disorders by regulating immune cell functions, such as differentiation and mediator secretion. However, the role of MBD2 in COPD remains unknown.MethodsMBD2 protein expression in lung tissues of patients with COPD and cigarette smoke (CS)-exposed mice were evaluated by Western blot and immunohistochemistry. The role of MBD2 in cigarette smoke extract (CSE)-induction of inflammatory mediator expression in the human bronchial epithelial (HBE) cell line was assessed by silencing MBD2 expression in vitro. The involvement of signaling pathways in mediation of inflammation was tested with signaling inhibitors.ResultsCompared with controls, MBD2 expression was distinctly reduced in the bronchial epithelium of both patients with COPD and CS-exposed mice. Moreover, MBD2 expression was decreased in HBE after CSE stimulation in vitro. Moreover, MBD2 knockdown enhanced interleukin (IL)-6 and IL-8 expression in HBE in the presence and absence of CSE treatment by the ERK signaling pathway.ConclusionMBD2 protein expression was reduced in the airway epithelium of COPD. In HBE, this reduced expression was associated with increased levels of IL-6 and IL-8 mediated by the ERK pathway. These results suggest that MBD2 could contribute to chronic airway inflammation in COPD.

MBD1 and MeCP2 expression in embryos and placentas from transgenic cloned goats.

DNA methylation is an important form of epigenetic regulation in mammalian development. Methyl-CpG-binding domain protein 1 (MBD1) and methyl-CpG-binding domain protein 2 (MeCP2) are two members of the MBD subfamily of proteins that bind methylated CpG to maintain the silencing effect of DNA methylation. Given their important roles in linking DNA methylation with gene silencing, this study characterized the coordinated mRNA expression and protein localization of MBD1 and MeCP2 in embryos and placentas and aimed to analysis the effects of MBD1 and MeCP2 on transgenic cloned goats. Our result showed that MBD1 expression of transgenic cloned embryo increased significantly at the 2-4-cell and 8-16-cell stages (P < 0.05), then decreased at the morula and blastocyst stages (P < 0.05); MeCP2 expression in transgenic cloned embryo was significant decreased at the 2-4-cell stage and increased at the 8-16-cell stage (P < 0.05). Placenta morphology analysis showed that the cotyledon number of deceased transgenic cloned group (DTCG) was significantly lower than that the normal goats (NG) and in the live transgenic cloned goats (LTCG) (P < 0.05). MBD1 and MeCP2 were clearly detectable in the placental trophoblastic binucleate cells by immunohistochemical staining. Moreover, MBD1 and MeCP2 expression in DTCG was significant higher than in the NG and the LTCG (P < 0.05). In summary, aberrant expression of methylation CpG binding proteins MBD1 and MeCP2 was detected in embryonic and placental development, which reflected abnormal transcription regulation and DNA methylation involved in MBD1 and MeCP2. These findings have implications in understanding the low efficiency of transgenic cloning.

MBD2 Regulates Th17 Cell Differentiation and Experimental Severe Asthma by Affecting IRF4 Expression.

Th17 cells and IL-17 participate in airway neutrophil infiltration characteristics in the pathogenesis of severe asthma. Methyl-CpG binding domain protein 2 (MBD2) expression increased in CD4+ T cells in peripheral blood samples of asthma patients. However, little is known about that epigenetic regulation of MBD2 in both immunological pathogenesis of experimental severe asthma and CD4+ T cell differentiation. Here, we established a neutrophil-predominant severe asthma model, which was characterized by airway hyperresponsiveness (AHR), BALF neutrophil granulocyte (NEU) increase, higher NEU and IL-17 protein levels, and more Th17 cell differentiation. In the model, MBD2 and IRF4 protein expression increased in the lung and spleen cells. Under overexpression or silencing of the MBD2 and IRF4 gene, the differentiation of Th17 cells and IL-17 secretion showed positive changes. IRF4 protein expression showed a positive change with overexpression or silencing of the MBD2 gene, whereas there was no significant difference in the expression of MBD2 under overexpression or silencing of the IRF4 gene. These data provide novel insights into epigenetic regulation of severe asthma.

Changes in poly(ADP-ribosyl)ation patterns in workers exposed to BTX.

Occupational exposure to (benzene, toluene and xylene, BTX is common in the Chinese workplace. Chronic occupational exposure to benzene is associated with an increased risk of hematological malignancies such as acute myeloid leukemia (AML), but the underlying mechanisms are still unclear. This study investigates changes in poly(ADP-ribosyl)ation and DNA methylation in subjects occupationally exposed to a BTX. Blood DNA samples and exposure data were obtained from subjects with different levels of exposure, including 132 decorators, 129 painters, and 130 unexposed referents in a container-manufacturing factory in Shenzhen, China. Occupational exposure assessment included personal monitoring of airborne benzene, toluene and xylene. Hematological parameters were measured and the cytokinesis-block micronucleus (CBMN) assay was used to detect DNA damage in peripheral lymphocytes. Quantitative real-time PCR was used to detect the mRNA expression of poly(ADP-ribose) polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG), DNA methyltransferases (DNMTs) including DNMT1, DNMT3a and DNMT3b, methyl-CpG-binding domain protein 2(MBD2). PARP1 assay was used to measure PARP activity. Airborne levels of benzene, toluene and xylene in the two exposed groups were significantly higher than those of controls (P<0.001). The two exposed groups (decorators, painters) showed decreased PARP1, DNMTs and MBD2 expression relative to controls (P<0.05), and PARP activity was also decreased (P<0.05). Decreased PARP1, DNMT1, DNMT3a, DNMT3b and MBD2 mRNA expression was correlated with increased airborne BTX (Pearson's r: −0.587, −0.314, −0.636, −0.567 and −0.592 respectively, P<0.001). No significant differences in hematological parameters and CBMN were found among the three groups. Together, these results suggest that decreased DNMTs, MBD2 and PARP1 might be involved in the global hypomethylation associated with BTX exposure, and the imbalance of PARP/PARG might participate in the down-regulation of DNMTs. This is the first human study to link altered poly(ADP-ribosyl)ation patterns, which reproduce the aberrant epigenetic patterns found in benzene-treated cells, to chronic occupational exposure to BTX.

Chronic cerebrovascular hypoperfusion affects global DNA methylation and histone acetylation in rat brain

DNA methylation and histone acetylation can be modified by various pathological or physiological factors such as hypoxia, thus influencing gene expression. In this study, we investigated the changes of global DNA methylation and histone acetylation and the related enzymes in rat brain after chronic cerebrovascular hypoperfusion by bilateral common carotid occlusion (2-VO) surgery. Colorimetric and immunohistochemistry staining were used to evaluate the global DNA methylation and histone acetylation levels, respectively. The expressions of DNA methyltransferase 1/3a (DNMT1/3a), methyl-CpG binding domain protein 2 (MBD2), histone deacetylase 3 (HDAC3) and acetyltransferase (HAT) were assessed by Western blot. We found that the level of global DNA methylation was decreased to 31.7% (P <0.01) of the sham-operated group at 10 days and increased by 30% (P <0.01) compared with the sham group at 90 days after 2-VO surgery. DNMT3a expression was down-regulated to 75.7% of the sham group, while MBD2 expression was up-regulated by 95% compared with sham group at 90 days after 2-VO. The histone H3 acetylation level was markedly decreased to 75.3% of the sham group at 10 days and 73.5% at 90 days after 2-VO, while no significant change was found for histone H4 acetylation. HDAC3 expression was markedly down-regulated to 36% of the sham group, whereas cAMP-response element binding protein expression was up-regulated by 33.6% compared with the sham group at 90 days after 2-VO. These results suggest that chronic cerebrovascular hypoperfusion influences global DNA methylation and histone acetylation levels through the related enzymes, and therefore might contribute to several neurodegenerative diseases.

Expression and inhibition of MBD2 in gastric cancer

As gastric cancer (GC) involves changes in gene expression, proteins that bind to methylated DNA, such as methyl-CpG-binding domain protein 2 (MBD2) may be a potential target for cancer treatment. We used immunohistochemistry and immunofluorescence to detect the MBD2 expression in GC tissues and SGC-7901 (GC cell line), RT-PCR and western blotting to evaluate the knockdown of MBD2 by specific siRNAs, and MTT assay to analyse the proliferation of SGC-7901 after the MBD2-siRNA transfection. We found GC expression of MBD2 protein was higher than in matched non-cancerous tissues, and the MBD2 expression was associated with the size and histological grade of GC tissues. MBD2 protein was distributed in the nucleus of GC cells, and the MBD2 mRNA and protein could be inhibited by a specific siRNA. Finally, after 96 h of the MBD2-siRNA transfection, the growth rate of SGC-7901 decreased compared with controls. Hence MBD2 may be an underlying target of gastric cancer treatment.

Expression of DNMTs and MBD2 in GIST

The aim of this study was to investigate the protein expression of DNA methyltransferases (DNMTs, including DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L) and methyl-CpG-binding domain protein 2 (MBD2) in gastrointestinal stromal tumor (GIST). Immunohistochemistry and western blot analysis were used to detect expression of DNMT and MBD2 in 15 pairs of adult GIST and matched non-tumor tissues. The protein expression of DNMT1, DNMT2, DNMT3B, DNMT3L and MBD2 was significantly higher in adult GISTs compared to the matched non-tumor tissues (P<0.05). However, no significant difference was detected in the protein expression of DNMT3A between tumor and non-tumor tissues (P>0.05). Associations between DNMT1 expression and mitotic index, DNMT3B expression and tumor size, as well as DNMT3L expression and Helicobacter pylori infection were detected in GISTs (P<0.05). In conclusion, GISTs exhibit a high protein expression of DNMT (with the exception of DNMT3A) and MBD2.

Hypomethylation mediated by decreased DNMTs involves in the activation of proto-oncogene MPL in TK6 cells treated with hydroquinone

Hydroquinone (HQ), one of the most important metabolites derived from benzene, is known to be associated with acute myelogenous leukemia (AML) risk, however, its carcinogenic mechanism remains unclear. In this study, the epigenetic mechanism of HQ exposure was investigated. We characterized the epigenomic response of TK6 cells to HQ exposure, and examined the mRNA expression of DNA methyltransferases (DNMTs) including DNMT1, DNMT3a and DNMT3b, methyl-CpG-binding domain protein 2 (MBD2) and six proto-oncogenes (MPL, RAF1, MYB, MYC, ERBB2 and BRAF). Compared to the control cells, HQ exposure (2.5, 5.0, 10.0 and 20.0μM for 48h) resulted in the decrease of DNMTs and MBD2 expression, the global hypomethylation and increase of MPL at mRNA level. Meanwhile, most of these changes were in dose-dependent manner. Moreover, inhibition of DNMTs induced by 5-aza-2′-deoxycytidine (5-AZA), an identified DNMT inhibitor, caused more induction of MPL expression at mRNA level compared to the HQ (10.0μM) pre-treated group. Furthermore, treatment of HQ potentially led to MPL itself hypomethylation (10.0 and 20.0μM reduced by 47% and 44%, respectively), further revealing that the activation of proto-oncogene MPL was related to hypomethylation in its DNA sequences. In conclusion, hypomethylation, including global and specific hypomethylation, might be involved in the activation of MPL, and the hypomethylation could be induced by decreased DNMTs in TK6 cells exposed to HQ.

MiR-373 Negatively Regulates Methyl-CpG-Binding Domain Protein 2 (MBD2) in Hilar Cholangiocarcinoma

microRNAs (miRNAs) are a class of non-coding, single-stranded RNA molecules that regulate gene expression at the posttranscriptional level. Methyl-CpG-binding domain proteins (MBPs) are transcription repressors through binding to methylated gene promoters. Recent studies have shown that the effect of miRNAs on DNA methylation by targeting DNA methyltransferase (DNMTs) and/or MBPs plays an important role in various human cancers. This study focuses on the regulation of MBPs by miR-373 and its downstream effect in hilar cholangiocarcinoma. miR-373 was investigated by TaqMan miRNA Assay; mRNA and protein of MBD1, MBD2, and Mecp2 were determined by QuantiTect(®) Primer Assays and Western blotting, respectively; RASSF1A mRNA was measured by SYBR-Green real-time PCR; The targeting at MBD2-3'UTR by miR-373 was evaluated by dual-luciferase reporter gene assay. miR-373 decreased and closely associated with poor cell differentiation, advanced clinical stage, and shorter survival in hilar cholangiocarcinoma; MBD2 exclusively over-expressed and reciprocally related to miR-373; precursor miR-373 inhibited the luciferase activity of MBD2-3'UTR construct; exogenous miR-373 suppressed the expression of MBD2 and enhanced RASSF1A mRNA in QBC(939) cells; anti-miR-373 inhibitor up-regulated the expression of MBD2 and reduced RASSF1A mRNA in HIBEpic cells. miR-373 is one negative regulator of MBD2. In hilar cholangiocarcinoma, down-expression of miR-373 leads to increase of MBD2, which in turn suppresses the methylation-mediated gene such as RASSF1A.

Hepatitis B virus X protein induces hypermethylation of p16INK4Apromoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis

The aim of the present study was to authenticate the involvement of DNA methyltransferases (DNMTs) and methyl-CpG binding domain protein 2 (MBD2) in the process of HBx induced p16(INK4A) promoter hypermethylation in HBV-related hepatocellular carcinoma (HCC) and their corresponding noncancerous liver tissues. Eighty-eight fresh tissue specimens of surgically resected HBV-associated HCC and their corresponding noncancerous liver tissues were studied. The methylation status of the p16(INK4A) promoter was determined by methylation-specific polymerase chain reaction (MSP). Reverse transcription and real-time polymerase chain reaction (RT-PCR) showed the expression of DNMTs, MBD2 and HBx. Western blot and immunohistochemistry were used for the protein analysis of HBx, DNMT1, DNMT3A and P16. Tissue HBV-DNA levels were determined by RT-PCR. HBV genotype was examined by nested PCR and restriction fragment length polymorphism (RFLP). In the corresponding noncancerous liver tissues, higher HBx expression was associated with the hypermethylation of the p16(INK4A) promoter. HBx was positively correlated with the DNMT1 and DNMT3A at both the mRNA and protein level. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negatively correlated with p16 protein expression. In HCC tissues, HBx was positively correlated with DNMT1 and DNMT3A at both mRNA and protein level, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression. The methylation status of the p16(INK4A) promoter did not correlate with clinicopathological characteristics. DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16(INK4A) promoter in the early stages of HBV-associated HCC. HBx-DNMTs-p16(INK4A) promoter hypermethylation may constitute a mechanism for tumorigenesis during HBV-associated hepatocarcinogenesis.

Promoter CpG island hypermethylation- and H3K9me3 and H3K27me3-mediated epigenetic silencing targets the deleted in colon cancer (DCC) gene in colorectal carcinogenesis without affecting neighboring genes on chromosomal region 18q21

Chromosomal loss of 18q21 is a frequent event in colorectal cancer (CRC) development, suggesting that this region harbors tumor suppressor genes (TSGs). Several candidate TSGs, among which methyl-CpG-binding domain protein 1 (MBD1), CpG-binding protein CXXC1, Sma- and Mad-related protein 4 (SMAD4), deleted in colon cancer (DCC) and methyl-CpG-binding domain protein 2 (MBD2) are closely linked on a 4-Mb DNA region on chromosome18q21. As TSGs can be epigenetically silenced, this study investigates whether MBD1, CXXC1, SMAD4, DCC and MBD2 are subject to epigenetic silencing in CRC. Methylation-specific polymerase chain reaction and sodium bisulfite sequencing of these genes show that DCC, but not MBD1, CXXC1, SMAD4 and MBD2, has promoter CpG island methylation in CRC cell lines and tissues {normal mucosa [29.5% (18/61)], adenomas [81.0% (47/58)] and carcinomas [82.7% (62/75)] (P = 8.6 x 10(-9))} that is associated with reduced DCC expression, independent of 18q21 loss analyzed by multiplex ligation-dependent probe amplification. Reduced gene expression of CXXC1, SMAD4 and MBD2 correlates with 18q21 loss in CRC cell lines (P = 0.04, 0.02 and 0.02, respectively). Treatment with the demethylating agent 5-aza-2'-deoxycytidine, but not with the histone deacetylase inhibitor trichostatin A exclusively restored DCC expression in CRC cell lines. Chromatin immunoprecipitation studies reveal that the DCC promoter is marked with repressive histone-tail marks H3K9me3 and H3K27me3, whereas activity related H3K4me3 was absent. Only active epigenetic marks were detected for MBD1, CXXC1, SMAD4 and MBD2. This study demonstrates specific epigenetic silencing of DCC in CRC as a focal process not affecting neighboring genes on chromosomal region 18q21.

DNA Methylation-Related Chromatin Modification in the Regulation of Mouse δ-Opioid Receptor Gene

DNA methylation plays critical roles in gene-silencing through chromatin modification. We reported previously that promoter-region CpG methylation repressed mouse delta-opioid receptor (mDOR) gene expression. In the current study, we demonstrated that the methylation of mDOR gene promoter is correlated with a repressive chromatin structure that has less HaeIII and MspI nuclear accessibility and more deacetylated histone H3 and H4 than that of unmethylated mDOR promoter. Chromatin immunoprecipitation analysis showed the association of a methyl-CpG-binding domain protein 2 (MBD2) with methylated mDOR promoter. Transient expression of MBD2 enhanced the repression of partially methylated mDOR promoter activity, and this repression was partially reversed by treatment of trichostatin A, a specific histone deacetylase inhibitor, indicating that MBD2 may mediate DNA methylation-related chromatin modification through recruiting histone deacetylases to mDOR promoter region. In addition, trichostatin A treatment increased both methylated mDOR promoter activity in a transient transfection assay and endogenous mDOR mRNA level in Neuro2A cells. Taken together, these results demonstrate that the mDOR gene expression is regulated by DNA methylation-related chromatin modification, especially histone acetylation and deacetylation.