Trace Elements Status and Metallothioneins DNA Methylation Influence Human Hepatocellular Carcinoma Survival Rate

BackgroundMechanisms underlying hepatocellular carcinoma (HCC) development are largely unknown. The role of trace elements and proteins regulating metal ions homeostasis, i.e. metallothioneins (MTs), recently gained an increased interest. Object of the study was to investigate the role of promoter DNA methylation in MTs transcriptional regulation and the possible prognostic significance of serum trace elements in HCC.MethodsForty-nine HCC patients were enrolled and clinically characterized. Cu, Se, and Zn contents were measured by Inductively Coupled Plasma Mass Spectrometry in the serum and, for a subset of 27 patients, in HCC and homologous non-neoplastic liver (N) tissues. MT1G and MT1H gene expression in hepatic tissues was assessed by Real-Time RT-PCR and the specific promoter DNA methylation by Bisulfite-Amplicon Sequencing.ResultsPatients with Cu serum concentration above the 80th percentile had a significantly decreased survival rate (P < 0.001) with a marked increased hazard ratio for mortality (HR 6.88 with 95% CI 2.60–18.23, P < 0.001). Se and Zn levels were significantly lower in HCC as compared to N tissues (P < 0.0001). MT1G and MT1H gene expression was significantly down-regulated in HCC as compared to N tissues (P < 0.05). MTs promoter was hypermethylated in 9 out of the 19 HCC tissues showing MTs down-regulation and methylation levels of three specific CpGs paralleled to an increased mortality rate among the 23 patients analyzed (P = 0.015).ConclusionsMT1G and MT1H act as potential tumor suppressor genes regulated through promoter DNA methylation and, together with serum Cu concentrations, be related to survival rate in HCC.

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Hypermethylation of MT1G and MT1H CpGs Promoter Sites and High Serum Levels of Cu Affect Survival Rate of Patients Affected by Hepatocellular Carcinoma

Current Developments in Nutrition ◽

10.1093/cdn/nzaa067_018 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1791-1791

Author(s):

Domenica De Santis ◽

Silvia Udali ◽

Andrea Ruzzenente ◽

Greta Beschin ◽

Patrizia Pattini ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Methylation ◽

Trace Elements ◽

Survival Rate ◽

Mortality Rate ◽

Liver Tissue ◽

Kaplan Meier ◽

Promoter Dna Methylation ◽

Promoter Dna

Abstract Objectives Recent evidences suggest a principal role of trace elements and metallothioneins (MTs), proteins involved in metal ions homeostasis and detoxification, in hepatocellular carcinogenesis. The study was designed to evaluate whether serum and liver tissue concentrations of the trace elements Cu, Zn and Se are implicated in survival rate of hepatocellular carcinoma (HCC) patients and if promoter DNA methylation is involved in trace elements-related proteins regulation. Methods Cu, Zn and Se levels were determined in serum and liver tissue samples, both HCC and homologous non neoplastic tissue (N) of 27 HCC patients by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Gene expression analysis of MT1G and MT1H, was performed by Real-time qPCR in HCC and N tissue. Promoter DNA methylation of a region overlapping MT1G and MT1H promoters was assessed by bisulfite amplicon sequencing (BSAS) in HCC and N tissues of 23 patients. Kaplan-Meier survival curves were drawn using the log-rank test (Mantel-Cox test) to examine the differences in survival according to serum trace elements and to gene-specific methylation levels. Results Kaplan-Meier analysis according to serum Cu levels showed that subjects within the highest quintile had an increased mortality rate (88.9%) compared with the other four quintiles (P = 0.025). Considering the 80th percentile of Cu levels (1118 μg/L), subjects with Cu concentrations above this value had a significantly decreased survival rate (P < 0.001). Se and Zn content were depleted in HCC tissues as compared to N tissues (P < 0.0001). MT1G and MT1H were strongly repressed in HCC tissues and precisely, MT1H in 24 out of 27 HCC tissues (P = 0.008) and MT1G in 23 out of 27 HCC tissues (P = 0.037). Nine out of 19 HCC tissues showing a down-regulation of MTs with three CpG sites, significantly hypermethylated in HCC tissue as compared to N tissue (P < 0.05). Considering the median methylation level, patients with higher methylation values showed increased mortality rate (P = 0.015). Conclusions The significant repression of MT1G and MT1H in HCC tissue is related to promoter hypermethylation and support the hypothesis of MT1G and MT1H as possible tumor suppressor genes in HCC. The evidence of promoter methylation levels and survival rate association provide new insights for the role of DNA methylation in liver carcinogenesis. Funding Sources N/A.

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Metallothioneins Gene Regulation Through Promoter DNA Methylation According to Zn and Cu Trace Elements Status in Human Hepatocellular Carcinoma (P05-002-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz030.p05-002-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Domenica De Santis ◽

Silvia Udali ◽

Filippo Mazzi ◽

Andrea Ruzzenente ◽

Greta Beschin ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Methylation ◽

Trace Elements ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Funding Sources ◽

Promoter Dna Methylation ◽

Serum Trace Elements ◽

Promoter Dna

Abstract Objectives Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, yet mechanisms of hepatocarcinogenesis are largely unknown. A particular interest was recently dedicated to the role of trace elements and metallothioneins (MTs), a group of proteins involved in metal ions homeostasis and detoxification, have been suggested as possible tumor suppressor genes. The study of MTs transcriptional regulation by promoter DNA methylation is the object of study as a possible mechanism responsible for gene silencing through epigenetics. Methods Twenty-seven HCC patients undergoing surgery intervention were enrolled and clinically characterized. MT1G and MT1H gene expression was performed by Real Time qPCR. DNA methylation analysis in 23 HCC and homologous non-neoplastic liver tissue (N) was performed by Bisulfite-Amplicon Sequencing (BSAS) in an overlapping region (∼400 bp) of the promoters of the two genes. Cu and Zn concentrations were measured in serum and liver tissues (HCC and N) by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Kaplan-Meier analysis of survival was performed according to serum trace elements. Results MT1G and MT1H were transcriptionally repressed in HCC tissue as compared to N. A correlation was observed between the mRNA levels of the two MTs, in particular MT1G was repressed in 23 out of 27 HCC tissue (P = 0.0366) and MT1H was repressed in 24 out of 27 HCC tissue (P = 0.0077). The promoter region resulted hypermethylated in 9 out of 19 HCC that showed MT1G and MT1H down-regulation. Serum Zn and Cu levels were within the normal range while HCC tissue exhibited significantly reduced Zn levels as compared to N (P < 0.0001). Tissue Cu levels did not show significant differences. Serum trace elements levels were also analyzed according to patients clinical features and those with Cu levels higher than the 75th percentile had a significantly poorer prognosis than those within the lowest Cu levels quartile (P < 0.05). Conclusions MT1G and MT1H are repressed in HCC tissue. In a subset of patients the downregulation was associated to promoter hypermethylation, supporting the hypothesis of MT1G and MT1H as possible tumor suppressor genes in HCC. Evidence of a correlation between serum Cu levels and survival rate provide new insights for the role of this microelement in liver carcinogenesis. Funding Sources No funding sources.

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Cellular Heterogeneity–Adjusted cLonal Methylation (CHALM) improves prediction of gene expression

Nature Communications ◽

10.1038/s41467-020-20492-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jianfeng Xu ◽

Jiejun Shi ◽

Xiaodong Cui ◽

Ya Cui ◽

Jingyi Jessica Li ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cellular Heterogeneity ◽

Biological Functions ◽

Global Correlation ◽

Differentially Methylated Genes ◽

Promoter Dna Methylation ◽

Functional Consequences ◽

Promoter Dna ◽

Underlying Mechanisms

AbstractPromoter DNA methylation is a well-established mechanism of transcription repression, though its global correlation with gene expression is weak. This weak correlation can be attributed to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity–Adjusted cLonal Methylation (CHALM) as a methylation quantification method. CHALM improves understanding of the functional consequences of DNA methylation, including its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method enables detection of differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

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Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation

Blood ◽

10.1182/blood-2007-01-068635 ◽

2007 ◽

Vol 110 (4) ◽

pp. 1343-1352 ◽

Cited By ~ 43

Author(s):

Rodwell Mabaera ◽

Christine A. Richardson ◽

Kristin Johnson ◽

Mei Hsu ◽

Steven Fiering ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Fetal Liver ◽

Globin Gene ◽

Erythroid Cells ◽

Promoter Dna Methylation ◽

Globin Promoter ◽

Promoter Dna

AbstractThe mechanisms underlying the human fetal-to-adult β-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human γ- and β-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at −162 of the γ promoter and −126 of the β promoter are hypomethylated in ABM and FL, respectively. We also studied γ-globin promoter methylation during in vitro differentiation of erythroid cells. The γ promoters are initially hypermethylated in CD34+ cells. The upstream γ promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient γ-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human γ- and β-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human β-globin locus gene switching.

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Large Sex Differences in Chicken Behavior and Brain Gene Expression Coincide with Few Differences in Promoter DNA-Methylation

PLoS ONE ◽

10.1371/journal.pone.0096376 ◽

2014 ◽

Vol 9 (4) ◽

pp. e96376 ◽

Cited By ~ 18

Author(s):

Daniel Nätt ◽

Beatrix Agnvall ◽

Per Jensen

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Sex Differences ◽

Brain Gene Expression ◽

Promoter Dna Methylation ◽

Promoter Dna

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Genome-wide repressive capacity of promoter DNA methylation is revealed through epigenomic manipulation

10.1101/381145 ◽

2018 ◽

Cited By ~ 8

Author(s):

Keegan Korthauer ◽

Rafael A. Irizarry

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Statistical Inference ◽

Transcriptional Repression ◽

Strong Association ◽

Large Body ◽

Strong Relationship ◽

Open Science ◽

Promoter Dna Methylation ◽

Promoter Dna

AbstractThe scientific community is increasingly embracing open science. This growing commitment to open science should be applauded and encouraged, especially when it occurs voluntarily and prior to peer review. Thanks to other researchers’ dedication to open science, we have had the privilege of conducting a reanalysis of a landmark experiment published as a preprint with data made available in a public repository. The study in question found that promoter DNA methylation is frequently insufficient to induce transcriptional repression, which appears to contradict a large body of observational studies showing a strong association between DNA methylation and gene expression. This study was the first to evaluate whether forcibly methylating thousands of DNA promoter regions is sufficient to suppress gene expression. The authors’ data analysis did not find a strong relationship between promoter methylation and transcriptional repression. However, their analyses did not make full use of statistical inference and applied a normalization technique that removes global differences that are representative of the actual biological system. Here we reanalyze the data with an approach that includes statistical inference of differentially methylated regions, as well as a normalization technique that accounts for global expression differences. We find that forced DNA methylation of thousands of promoters overwhelmingly represses gene expression. In addition, we show that complementary epigenetic marks of active transcription are reduced as a result of DNA methylation. Finally, by studying whether these associations are sensitive to the CG density of promoters, we find no substantial differences in the association between promoters with and without a CG island. The code needed to reproduce are analysis is included in the public GitHub repository github.com/kdkorthauer/repressivecapacity.

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