Abstract
BackgroundAberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current study to validate our previous microarray data and to identify novel hypermethylated gene promoters.MethodsThe validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour tissues from 30 patients with CRC.ResultsData from our previous genome search have received confirmation in the new set of 8 patients with CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated.ConclusionThe findings highlight the usefulness of combining gene expression patterns and epigenetic data to identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being associated with a more advanced stage.
Highlights
Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease
Results Quantitative Real Time PCR (qPCR) validation of deregulated genes Among the genes higlighted as significantly deregulated in our previous microarray study [14], 24 genes were selected for further validation by using the quantitative Real-Time PCR, based on their cellular function, such as transport, signal transduction, intracellular and cell surface signalling, cell cycle, replication-repair of DNA, and protein folding. (Table 3)
QPCR assay was applied to analyse mRNA expression of the 24 selected genes, as well as of three control housekeeping genes (GAPDH, EEF1A1 and HRPT1) in the tumour and normal tissues taken from new cohort of 8 patients with Colorectal cancer (CRC)
Summary
Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. The second event is represented by local DNA hypermethylation of CpG islands, short sequences rich in CpG dinucleotides in the 5'untranscribed region (5'-UTR) This event occurs in approximately half of all human genes [9,10] silences specific TSG, and accelerates cancer formation [11,12]. Cancer-specific promoter methylation may by itself serve as a valuable clue to uncover novel TSG
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