Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma

Transcriptional silencing of tumor suppressor genes and other cancer-related genes induced by promoter CpG island hypermethylation is an important epigenetic mechanism of hepatocarcinogenesis. Previous studies have established methylation profiles of hepatocellular carcinomas (HCCs) and demonstrated that methylation of several candidate genes in resected tissues may be associated with time to recurrence. The goals of our study were to test whether specific promoter methylation and mRNA levels of candidate genes, as well as global changes in DNA methylation, can be linked with time to recurrence and clinicopathological variables in a homogenous study group of HCC patients. Forty-three tumorous and 45 non-tumorous liver tissue samples from the surgical margin were obtained from HCV-positive, HBV-negative HCC patients who underwent tumor resection surgery and who were monitored for tumor recurrence thereafter (median follow-up time: 16 months (range, 0 – 79 months)). Methylation-specific PCR was used to assess the promoter methylation status of P16(INK4a), SOCS-1, RASSF1A, APC, GSTP1, RIZ1, and MGMT genes, while the level of LINE-1 methylation was used as marker of global DNA methylation levels. Methylation frequencies in P16(INK4a), RASSF1A, APC, GSTP1, and RIZ1 genes were significantly greater in tumorous versus non-tumorous tissues. Methylation of RIZ1 in non-tumorous tissues was significantly associated with time to recurrence. Additionally, genomic DNA was significantly more hypomethylated in tumorous tissues, and this change was associated with shorter recurrence, but not with clinicopathological features. In conclusion, this study supports the role of aberrant methylation in the pathobiology of HCV-positive HCCs. The finding that RIZ1 methylation and increased levels of LINE-1 hypomethylation in non-tumorous tissues are associated with time to recurrence underscores the importance of assessing the epigenetic state of the liver remnant

Oxidative DNA damage in the cerebellum of BTBR mice.

<p>(<b>A</b>) Levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 8-oxodG in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals (mean ± SD, n = 13). (<b>C</b>) The extent of mitochondrial DNA damage in the cerebellum of BTBR T+tf/J mice (mean ± SD, n = 5). (<b>D</b>) The expression of base excision DNA repair genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice. The expression of <i>Ogg1, Ung, Apex1</i>, and <i>Polβ</i> genes was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5). (<b>E</b>) Correlation plots of the expression of <i>Ogg1</i> and the 8-oxodG level in mouse genomic DNA. Each symbol represents an individual animal.</p

Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

<div><p>The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (<i>Ogg1</i>) and increased expression of <i>de novo</i> DNA methyltransferases 3a and 3b (<i>Dnmt3a</i> and <i>Dnmt3b</i>). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression <i>1 (Tet1)</i> and <i>Tet2</i> genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of <i>OGG1</i> alterations in pathogenesis of autism.</p></div

Levels of 5mC and 5hmC in cerebellar genomic DNA.

<p>(<b>A</b>) Levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 5mC and 5hmC in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals. Values were represented as open and closed circles as well as box plots. * - Significantly different from C57BL/6J mice or autism-free individuals. (<b>C</b>) Correlation plots of the 5hmC and the 8-oxo-7-hydrodeoxyguanosine (8-oxodG) levels in mouse and human cerebellar genomic DNA.</p

Western blot analysis of histone H3K4, H3K9, H3K27, and H4K20 trimethylation; H3K9, H4K20 dimethylation and H3K9, H3K27, and H4K16 acetylation in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>Densitometric analysis of the immunostaining results is shown as percent change in histone modification level in the cerebellum BTBR T+tf/J mice relative to the corresponding values in C57BL/6J mice, which was assigned a value of 100% (mean ± SD, n = 5).</p

Whole genome microarray analysis of gene expression in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>(<b>A</b>) Heat map illustrating significant differences in global gene expression between BTBR T+tf/J and C57BL/6J mice. The color bar identifies high-expressed (red) and low-expressed (green) genes. (<b>B</b>) Principal component analysis illustrating similarities and differences between BTBR T+tf/J and C57BL/6J mouse strains. (<b>C</b>) Venn diagram showing genes that were significantly different between BTBR T+tf/J and C57BL/6J mice. (<b>D</b>) Summary of molecular pathways that significantly differ between BTBR T+tf/J and C57BL/6J mice. The Ingenuity Pathway Analysis database (version 9.0) was used to determine and visualize molecular pathways enriched by the significant mRNA transcripts a P-value of <0.05 were considered “enriched”.</p

The expression of chromatin-modifying (A) and one carbon metabolism (B) genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>The gene expression was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5).</p