RESUMO
AIM: Methylation status of genome varies between pre-acute-on-chronic hepatitis B liver failure (pre-ACHBLF), acute-on-chronic hepatitis B liver failure (ACHBLF), and chronic hepatitis B patients. This study aimed to find better prognostic indicators for acute-on-chronic liver failure. METHODS: The level of global genome methylation in peripheral blood mononuclear cells (PBMCs) was detected. The overall genome methylation rate was determined using MethylFlash™ Methylated DNA Quantification Kit(Colorimetric). DNMT activity were measured using DNA Methyltransferase Activity/Inhibition Assay Kit. Gene expression of DNA methyltransferases (DNMT)ï¼methyl-CpG-binding domain (MBD) were detected by qRT-PCR. RESULTS: The global genome methylation level in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). There was also obvious difference of the global genome methylation level between pre-ACHBLF group and CHB group (P<0.001). Meanwhile, the activity of DNMT in ACHBLF group was significantly higher than that in chronic hepatitis B group (P<0.001). The mRNA expression level of DNMT1 was higher than that in pre-ACHBLF group (P<0.01) and CHB group (PP<0.001). The mRNA expression level of MBD1 in ACHBLF group was also higher than that in CHB group (P<0.001) and healthy controls (HCs) (P<0.01). And the mRNA expression level of MBD3 and MBD4 in ACHBLF, pre-ACHBLF and CHB group were lower than that in HCs (P<0.001). Meanwhile we observed an opposite change in the mRNA expression level of MECP2. The ROC curve suggested that global genome methylation level was a better prognostic predictor than MELD score in ACHBLF (AUC 0.950, SE 0.0237, 95%CI 0.874-0.986 VS AUC 0.863, SE 0.0439, 95%CI 0.765-0.931, P=0.0429). CONCLUSIONS: Genome methylation level can be a good biomarker in predicting the severity and prognosis of ACHBLF.
Assuntos
Insuficiência Hepática Crônica Agudizada , Hepatite B Crônica , Humanos , Prognóstico , Insuficiência Hepática Crônica Agudizada/genética , Hepatite B Crônica/complicações , Hepatite B Crônica/genética , Leucócitos Mononucleares , Metilação de DNA/genética , RNA Mensageiro/análise , DNARESUMO
SETDB1 and SETDB2 mediate trimethylation of histone H3 lysine 9 (H3K9), an epigenetic hallmark of repressive chromatin. They contain a non-canonical methyl-CpG-binding domain (MBD) and bifurcated SET domain, implying interplay between H3K9 trimethylation and DNA methylation in SETDB functions. Here, we report the crystal structure of human SETDB2 MBD bound to the cysteine-rich domain of a zinc-binding protein, C11orf46. SETDB2 MBD comprises the conserved MBD core and a unique N-terminal extension. Although the MBD core has the conserved basic concave surface for DNA binding, it utilizes it for recognition of the cysteine-rich domain of C11orf46. This interaction involves the conserved arginine finger motif and the unique N-terminal extension of SETDB2 MBD, with a contribution from intermolecular ß-sheet formation. Thus, the non-canonical MBD of SETDB1/2 seems to have lost methylated DNA-binding ability but gained a protein-protein interaction surface. Our findings provide insight into the molecular assembly of SETDB-associated repression complexes.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição , Humanos , Cisteína/metabolismo , DNA/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/química , Fatores de Transcrição/metabolismoRESUMO
Rett Syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants in the MECP2 gene. While the majority of RTT-causing variants are clustered in the methyl-CpG binding domain and NCoR/SMRT interaction domain, we report a female patient with a functionally uncharacterized MECP2 variant in the C-terminal domain, c.1030C>T (R344W). We functionally characterized MECP2-R344W in terms of protein stability, NCoR/SMRT complex interaction, and protein nuclear localization in vitro. MECP2-R344W cells showed an increased protein degradation rate without significant change in NCoR/SMRT complex interaction and nuclear localization pattern, suggesting that enhanced MECP2 degradation is sufficient to cause a Rett Syndrome-like phenotype. This study highlights the pathogenicity of the C-terminal domain in Rett Syndrome, and demonstrates the potential of targeting MECP2 protein stability as a therapeutic approach.
RESUMO
Intrinsically disordered proteins (IDPs) are multifunctional due to their ability to adopt different structures depending on the local conditions. The intrinsically disordered regions of methyl-CpG-binding domain (MBD) proteins play important roles in regulating growth and development by interpreting DNA methylation patterns. However, whether MBDs have a stress-protective function is far from clear. In this paper, soybean GmMBD10c protein, which contains an MBD and is conserved in Leguminosae, was predicted to be located in the nucleus. It was found to be partially disordered by bioinformatic prediction, circular dichroism and a nuclear magnetic resonance spectral analysis. The enzyme activity assay and SDS-PAGE results showed that GmMBD10c can protect lactate dehydrogenase and a broad range of other proteins from misfolding and aggregation induced by the freeze-thaw process and heat stress, respectively. Furthermore, overexpression of GmMBD10c enhanced the salt tolerance of Escherichia coli. These data validate that GmMBD10c is a moonlighting protein with multiple functions.
Assuntos
Glycine max , Proteínas Intrinsicamente Desordenadas , Glycine max/genética , Glycine max/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Metilação de DNA , L-Lactato Desidrogenase/metabolismoRESUMO
Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.
Assuntos
Bombyx , Proteínas de Ligação a DNA , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Bombyx/genética , Bombyx/metabolismo , Ilhas de CpG , Conformação Proteica em Folha beta , Metilação de DNA , GenômicaRESUMO
Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Ilhas de CpG , DNA/genética , Oligonucleotídeos , Luciferases/genéticaRESUMO
Background: Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients. Methods: Peripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity. Results: Compared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response. Conclusion: DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.
Assuntos
Artrite Reumatoide , Interleucina-6 , Animais , Camundongos , Artrite Reumatoide/tratamento farmacológico , DNA/uso terapêutico , Metilação de DNA , Interleucina-6/genética , Linfócitos T Reguladores , Linfócitos T CD4-Positivos/imunologiaRESUMO
Chronic Non-Communicable Diseases (NCDs) have been considered a global health problem, characterized as diseases of multiple factors, which are developed throughout life, and regardless of genetics as a risk factor of important relevance, the increase in mortality attributed to the disease to environmental factors and the lifestyle one leads. Although the reactive species (ROS/RNS) are necessary for several physiological processes, their overproduction is directly related to the pathogenesis and aggravation of NCDs. In contrast, dietary polyphenols have been widely associated with minimizing oxidative stress and inflammation. In addition to their antioxidant power, polyphenols have also drawn attention for being able to modulate both gene expression and modify epigenetic alterations, suggesting an essential involvement in the prevention and/or development of some pathologies. Therefore, this review briefly explained the mechanisms in the development of some NCDs, followed by a summary of some evidence related to the interaction of polyphenols in oxidative stress, as well as the modulation of epigenetic mechanisms involved in the management of NCDs.
RESUMO
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.
RESUMO
Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Ilhas de CpG , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência de Energia , Luciferases/metabolismo , Luciferases de Vaga-Lume/genéticaRESUMO
Methyl-CPG-Binding Domain (MBD) proteins play important roles in plant growth, development, and stress responses. The present study characterized the MBD families in watermelon and other cucurbit plants regarding the gene numbers and structures, phylogenetic and syntenic relationships, evolution events, and conserved domain organization of the MBD proteins. The watermelon ClMBD proteins were found to be localized in nucleus, and ClMBD2 and ClMBD3 interacted with ClIDM2 and ClIDM3. ClMBD2 bound to DNA harboring methylated CG sites but not to DNA with methylated CHG and CHH sites in vitro. The ClMBD genes exhibited distinct expression patterns in watermelon plants after SA and MeJA treatment and after infection by fungal pathogens Fusarium oxysporum f.sp. niveum and Didymella bryoniae. Overexpression of ClMBD2, ClMBD3, or ClMBD5 in Arabidopsis resulted in attenuated resistance against Botrytis cinerea, accompanied by down-regulated expression of AtPDF1.2 and increased accumulation of H2O2 upon B. cinerea infection. Overexpression of ClMBD1 and ClMBD2 led to down-regulated expression of AtPR1 and decreased resistance while overexpression of ClMBD5 resulted in up-regulated expression of AtPR1 and increased resistance against Pseudomonas syringae pv. tomato DC3000. Transcriptome analysis revealed that overexpression of ClMBD2 in Arabidopsis up-regulated the expression of a small set of genes that negatively regulate Arabidopsis immunity. These data suggest the importance of some ClMBD genes in plant immunity and provide the possibility to improve plant immunity through modification of specific ClMBD genes.
RESUMO
Cell-free DNA(cfDNA) methylation profiling is considered promising and potentially reliable for liquid biopsy to study progress of diseases and develop reliable and consistent diagnostic and prognostic biomarkers. There are several different mechanisms responsible for the release of cfDNA in blood plasma, and henceforth it can provide information regarding dynamic changes in the human body. Due to the fragmented nature, low concentration of cfDNA, and high background noise, there are several challenges in its analysis for regular use in diagnosis of cancer. Such challenges in the analysis of the methylation profile of cfDNA are further aggravated due to heterogeneity, biomarker sensitivity, platform biases, and batch effects. This review delineates the origin of cfDNA methylation, its profiling, and associated computational problems in analysis for diagnosis. Here we also contemplate upon the multi-marker approach to handle the scenario of cancer heterogeneity and explore the utility of markers for 5hmC based cfDNA methylation pattern. Further, we provide a critical overview of deconvolution and machine learning methods for cfDNA methylation analysis. Our review of current methods reveals the potential for further improvement in analysis strategies for detecting early cancer using cfDNA methylation.
RESUMO
Methylation changes at cytosine-guanine dinucleotide (CpG) sites in genes are closely related to cancer development. Thus, detection and quantification of low-abundance methylated DNA is critical for early diagnosis. Here, we report an atomic force microscopy (AFM)-based quantification method for DNA that contains methyl-CpG at a specific site, without any treatment to the target DNA such as chemical labeling, fluorescence tagging, or amplification. We employed AFM-tip-tethered methyl-CpG-binding proteins to probe surface-captured methylated DNA. We observed a linear correlation (R2 = 0.982) between the input copy number and detected copy number, in the low copy number regime (10 or fewer; subattomolar concentrations). For a mixture of methylated and nonmethylated DNA that resembles clinical samples, we were still able to quantify the methylated DNA. These results highlight the potential of our force-mapping-based quantification method for wide applications in early detection of diseases associated with methylated DNA.
Assuntos
Metilação de DNA , DNA , Ilhas de CpG , DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
DNA methylation alters the expression of certain genes without any alteration to the DNA sequence and is a dynamic process during normal hematopoietic differentiation. As an epigenetic regulator, methyl-CpG-binding domain protein 2 (MBD2) is an important member of the MBD protein family and is acknowledged as a "reader" of DNA methylation. We used a mouse model to study the effects of MBD2 on the early development of T cells. Here, we found that MBD2 deficiency led to retardation of T cell differentiation at the DN3 stage. Meanwhile, decreased proliferative capacity and increased apoptosis were detected in Mbd2-/- DN thymocytes. Furthermore, we found the WNT pathway was significantly down-regulated in Mbd2-/- DN thymocytes: DKK1 (Dickkopf-1) expression was significantly increased, while TCF7 (transcription factor 7) and c-MYC were down-regulated. Thus, these findings established that MBD2 acted as a dominant regulator to imprint DN T cell development via the WNT pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Linfócitos T/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Via de Sinalização WntRESUMO
Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.
RESUMO
Propofol (2,6-diisopropylphenol) is one of the most commonly used intravenous anesthetics and possesses a number of non-anesthetic effects, including antitumor function. The aim of the present study was to elucidate the antitumor molecular mechanism of propofol on A549 and H1299 cells. A549 and H1299 cells were treated in the presence or absence of different concentrations (0, 60 or 120 µmol) of propofol for different durations (0, 24, 48 or 72 h), and proliferation was detected by MTT and colony formation assays; the protein levels of optineurin (OPTN) ubiquitination, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), methyl-CpG binding domain protein 3 (MBD3) and Microtubule-associated protein 1A/1B-light chain 3 were detected by immunoblotting or quantitative (q)PCR; the methylation state of the HACE1 gene promoter was detected by bisulfite DNA sequencing; and binding of MBD3 on HACE1 gene promoter was detected by chromatin immunoprecipitation-qPCR. Propofol inhibited proliferation of A549 and H1299 cells and promoted HACE1-OPTN axis-mediated selective autophagy activity by increasing the protein expression levels of HACE1 via demethylating its promoter region. Furthermore, propofol promoted expression levels of MBD3 and binding to the -1,000 to -1 bp (transcription start site) region of HACE1 gene promoter. MBD3-knockdown experiments indicated that propofol inhibited proliferation of A549 cells in a MBD3-dependent manner. Thus, the findings of the present study provided a potential antitumor molecular mechanism mediated by propofol.
RESUMO
Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to loss of key cellular pathways in tumorigenesis. Methyl-CpG-targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA-demethylating agents. Microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in 12 pancreatic ductal adenocarcinoma (PDAC) cell lines. Among these, we focused on IRX4 (Iroquois homeobox 4) because IRX4 is located at chromosome 5p15.33 where PDAC susceptibility loci have been identified through genome-wide association study. IRX4 was greatly downregulated in all of the analyzed 12 PDAC cell lines by promoter hypermethylation. In addition, the IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected PDACs (18/28: 64%). Reexpression of IRX4 inhibited colony formation and proliferation in two PDAC cell lines, PK-1 and PK-9. In contrast, knockdown of IRX4 accelerated cell proliferation in an IRX4-expressing normal pancreatic ductal epithelial cell line, HPDE-1. Because IRX4 is a sequence-specific transcription factor, downstream molecules of IRX4 were pursued by microarray analyses utilizing tetracycline-mediated IRX4 inducible PK-1 and PK-9 cells; CRYAB, CD69, and IL32 were identified as IRX4 downstream candidate genes. Forced expression of these genes suppressed colony formation abilities for both PK-1 and PK-9. These results suggest that DNA methylation-mediated silencing of IRX4 contributes to pancreatic tumorigenesis through aberrant transcriptional regulation of several cancer-related genes.
Assuntos
Carcinoma Ductal Pancreático/genética , Proliferação de Células/genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Técnicas de Silenciamento de Genes/métodos , Proteínas de Homeodomínio/metabolismo , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Plasmídeos , Análise Serial de Proteínas , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , Neoplasias PancreáticasRESUMO
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.
RESUMO
MeCP2 is an essential transcriptional repressor that mediates transcriptional inhibition by binding to methylated DNA. The binding specificity of MeCP2 protein to methylated DNA was considered to depend on its methyl-CpG binding domain (MBD). In this study, we used atomic force microscope based single-molecular force spectroscopy to investigate the interaction of MeCP2 MBD and methylated DNA. The specific interaction forces of the MeCP2 MBD-methylated DNA complexes were measured for the first time. The dynamics was also investigated by measuring the unbinding force of the complex at different loading rates. In addition, the distribution of unbinding forces and binding probabilities of MeCP2 MBD and different DNA were studied at the same loading rate. It was found that MeCP2 MBD had weak interaction with hemi-methylated and unmethylated DNA compared to methylated DNA. This work revealed the binding characteristics of MeCP2 MBD and methylated DNA at the single-molecule level. It provides a new idea for exploring the molecular mechanism of MeCP2 in regulating methylation signals.
Assuntos
DNA/química , Proteína 2 de Ligação a Metil-CpG/química , Imagem Individual de Molécula , Metilação de DNA , Estrutura MolecularRESUMO
Uveal melanoma (UM) is a rare intraocular cancer with the highest incidence in northern latitudes. Metastases develop in approximately 50% of patients, whereafter the median survival is 13 months. Generally, the mutation burden of these tumors is low. Germline variants predisposing to UM have been previously described in BRCA1-associated protein 1 (BAP1). Recently, germline and somatic loss-of-function (LOF) variants in the methyl-CpG-binding domain 4 (MBD4) gene have been found to cause a hypermutated UM, and MBD4 also has been put forward as a gene predisposing to UM. We sequenced for MBD4 germline variants in 440 Finnish patients with UM and identified seven rare exonic missense variants in 16 (3.6%) patients, of which one likely alters MBD4 function. The frequency of likely pathogenic variants in our cohort is 0.23% (1/432; 95% CI, 0.01-1.28). We identified no LOF variants though their frequency in the Finnish population is 0.052%. Thus, our data do not support the suggestion that MBD4 germline variants predispose to UM. Somatic loss of MBD4 might modify the mutational burden in UM and change its response to immune checkpoint inhibitors.