La Croix du Nord : supplément régional à la Croix de Paris ["puis" grand journal quotidien du Nord de la France]

30 août 19191919/08/30 (A30,N8987).Appartient à l’ensemble documentaire : NordPdeC

Loss of Imprinting and Allelic Switching at the <em>DLK1-MEG3</em> Locus in Human Hepatocellular Carcinoma

<div><p>Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the <em>DLK1-MEG3</em> locus is frequently deregulated in HCC. Deregulation of <em>DLK1</em> and <em>MEG3</em> expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the <em>DLK1-MEG3</em> locus correlates linearly with global loss of DNA methylation in HCC (r² = 0.63, p<0.0001). Inhibition of DNMT1 in HCC cells using siRNA led to a reduction in <em>MEG3-DMR</em> methylation and concomitant increase in <em>MEG3</em> RNA expression. Allele-specific expression analysis identified loss of imprinting in 10 out of 31 informative samples (32%), rendering it one of the most frequent molecular defects in human HCC. In 2 cases unequivocal gain of bi-allelic expression accompanied by substantial loss of methylation at the <em>IG-DMR</em> could be demonstrated. In 8 cases the tumour cells displayed allelic switching by mono-allelic expression of the normally imprinted allele. Allelic switching was accompanied by gains or losses of DNA methylation primarily at <em>IG-DMR1</em>. Analysis of 10 hepatocellular adenomas (HCA) and 5 cases of focal nodular hyperplasia (FNH) confirmed that this epigenetic instability is specifically associated with the process of malignant transformation and not linked to increased proliferation <em>per se</em>. This widespread imprint instability in human HCC has to be considered in order to minimize unwanted side-effects of therapeutic approaches targeting the DNA methylation machinery. It might also serve in the future as predictive biomarker and for monitoring response to epigenetic therapy.</p> </div

Additional file 5: Figure S4. of Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival

DNA methylation analysis at imprinted loci in HCA, FNH, the adjacent liver tissues, and healthy liver samples

Additional file 2: Figure S1. of Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival

Global DNA methylation levels (measured by LINE1 methylation)

Relationship between DNA methylation and gene expression at the <i>DLK1-MEG3</i> locus.

<p>Primary HCC samples are defined as “hypermethylated” and “hypomethylated” using mean ±2×SD of the methylation levels of the adjacent non-cancerous liver tissues. Relative <i>MEG3</i> and <i>DLK1</i> expression is then compared among hyper-, normo-, and hypo-methylated subgroups. The horizontal lines inside the box represents the median of relative <i>MEG3</i> expression and the upper and lower edges of each box represent the 75th and 25th percentile respectively, while the bars denote the highest and lowest relative expression measured. * = 0.001</p

Allele-specific expression analysis of the <i>DLK1-MEG3</i> locus in human HCC.

<p>Quantitative SNP analysis was performed using pyrosequencing at a C/G polymorphism in exon 3, at SNP Rs8013873 for <i>MEG</i>3 and SNP Rs1802710 for <i>DLK</i>1. In tumour specimens showing genomic DNA polymorphisms, further SNP analysis was performed with the cDNAs from the same sample as well as with genomic and cDNA from the corresponding adjacent non-cancerous liver tissues. (A) Pyrosequencing analysis of the cDNA at SNP Rs8013873 shows mono-allelic <i>MEG</i>3 expression in HCC specimen #8 and bi-allelic expression in HCC specimen #21. (B) Pyrosequencing analysis of the cDNA at SNP Rs1802710 shows mono-allelic <i>DLK</i>1 expression in HCC specimen #33 and bi-allelic expression in HCC specimen #20. (C) <i>MEG</i>3 allelic switching is shown in case #25. Informative polymorphisms are found in the genomic DNAs of both tumour and the adjacent non-cancerous tissues and T/T expression in cDNA tumour but C/C expression in cDNA adjacent liver tissues. (D) <i>DLK</i>1 allelic switching is shown in case #30 with informative polymorphisms in the genomic DNAs of both tumour and the adjacent non-cancerous tissues and C/C expression in tumour cDNA but T/T expression in cDNA from the adjacent liver tissues. The confirmation of all genotyping results by Sanger sequencing is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049462#pone.0049462.s010" target="_blank">Figure S5</a>.</p

Additional file 3: Figure S2. of Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival

Cluster analysis of methylation patterns at imprinted loci in HCC using Illumina 27K methylation array from (A) Heidelberg n = 63) [23] and (B) Taiwanese cohort (n = 62) [24] shows subgrouping of HCC into three groups: hypermethylation and moderate and severe hypomethylation

DNA methylation analysis of the <i>IG-DMR1</i> of the <i>DLK1-MEG3</i> locus in human HCC.

<p>A) Gain of bi-allelic expression is accompanied by substantial loss of methylation. B) Tumour samples displaying retention of the original imprinting pattern by strict mono-allelic expression of the same allele as in the adjacent non-cancerous liver tissue show only minor variations in DNA methylation. C) Tumour samples demonstrating allelic switching (i.e., mono-allelic expression from the normally silenced imprinted allele) show variable changes of DNA methylation patterns to an intermediate degree. The numbers in the lower left corner of each bisulfite sequencing panel are the methylation values for the region according to pyrosequencing. “G” and “A” denote the sequence at SNP Rs12437020, which allows in heterozygous samples the differentiation of the two alleles. For the calculation of the statistical significance of the changes in DNA methylation the methylation level for each clone was calculated by computing the fraction of methylated CpG sites. From these values a mean methylation level of the tumor and adjacent liver tissue sample, respectively, was calculated. These two mean values were compared using Mann-Whitney-U-test and the p-values are depicted below the schematic representation of the bisulfite sequencing results.</p

Schematic representation of the <i>DLK1-MEG3</i> imprinted locus on chromosome 14q32.

<p>Maternally expressed genes are depicted in black and paternally expressed genes in grey. The intergenic differentially methylated region (<i>IG-DMR</i>) is located approximately 14 kb upstream to the first exon of <i>MEG3</i>. Studies in patients with UPD chromosome 14 revealed two regions with differential methylation at IG-DMR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049462#pone.0049462-Kagami2" target="_blank">[30]</a>. Therefore two pyrosequencing assays were developed at this region (<i>IG-DMR</i>1 and 2). In addition, three pyrosequencing assays were designed for one CpG island and two CTCF binding sites within the <i>MEG3</i>-DMR which was found to display parent-of-origin specific methylation (<i>MEG3</i>-DMR1, 2, and 3). Horizontal bar represents scale of 100 kbp.</p

Expression and DNA methylation of <i>MEG3</i> RNA and <i>DLK1</i> mRNA in primary human HCC.

<p><i>MEG3</i> RNA (A) and <i>DLK</i>1 mRNA (B) expression was measured in a series of 34 paired HCC using quantitative RT-PCR. For every sample, the RNA and mRNA levels were normalized to the mean expression of <i>GUSB</i> and <i>GAPDH</i>. Expression in primary HCC samples was then normalized to the corresponding adjacent non-cancerous liver tissues. Relative expression is presented as fold change in a logarithmic scale. Samples were classified as “up-regulated” if the lower limit of the 98% confidence interval was larger than 1 and “down-regulated” if the upper limit was smaller than 1. DNA methylation levels were measured using high-resolution quantitative pyrosequencing and are displayed below the expression data as hypermethylated (black box), hypomethylated (light grey) or normomethylated (white box). For definition of thresholds see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049462#s4" target="_blank">Materials and Methods</a>. The computed methylation levels are the average of two independent pyrosequencing runs. The pyrosequencing assays for <i>IG-DMR</i>1, <i>IG-DMR</i>2, and <i>MEG3-DMR</i>1 contain 5 CG sites, for <i>MEG3-DMR</i>2 9 CG sites, for <i>MEG3-DMR3</i> 6 CG sites. A positive association of high methylation with reduced <i>MEG3</i> expression (panel A), left part) and reduced methylation and low <i>DLK1</i> expression (panel B), left part) is clearly visible.</p