Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1Cin pancreatic endocrine tumorsReportar como inadecuado

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BMC Cancer

, 11:351

Genetics, genomics and epigenetics


BackgroundRASSF1A gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor PET but RASSF1A expression has never been studied. The RASSF1 locus contains two CpG islands A and C and generates seven transcripts RASSF1A-RASSF1G by differential promoter usage and alternative splicing.

MethodsWe studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the i methylation status of the RASSF1 CpG islands using methylation-specific PCR and pyrosequencing and ii expression of RASSF1 isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR MSP and by quantitative methylation-specific PCR qMSP; pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches.

ResultsMSP detected methylation in 16-20 80% PETs and 13-20 65% normal pancreas. At qMSP, 11-20 PETs 55% and 9-20 45% normals were methylated in at least 20% of RASSF1A alleles.

Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15-20 75% cases P = 0.01. The evaluation of mRNA expression of RASSF1 variants showed that: i RASSF1A was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET P = 0.003; ii RASSF1A methylation inversely correlated with its expression; iii RASSF1 isoforms were rarely found, except for RASSF1B that was always expressed and RASSF1C whose expression was 11.4 times higher in PET than in normal tissue P = 0.001. A correlation between RASSF1A expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in RASSF1A expression upon demethylating treatment.

ConclusionsRASSF1A gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm. RASSF1A is always expressed in PET and normal pancreas and its levels are inversely correlated with gene methylation. Isoform RASSF1C is overexpressed in PET and the recent demonstration of its involvement in the regulation of the Wnt pathway points to a potential pathogenetic role in tumor development.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-11-351 contains supplementary material, which is available to authorized users.

Giorgio Malpeli contributed equally to this work.

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Autor: Giorgio Malpeli - Eliana Amato - Mario Dandrea - Caterina Fumagalli - Valentina Debattisti - Letizia Boninsegna - Giuseppe 


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