Carcinogenesis is a complex event characterized by the progressive development of genetic and epigenetic aberrations which ultimately result in loss of physiological control of cell growth and differentiation. The two most important epigenetic mechanism are represented by the DNA methylation, the conversion of cytosine into methyl-cytosine catalyzed by the DNA methyltransferase And histone modifications . Changes in the DNA methylation pattern may occur everywhere in the DNA molecule. Global DNA hypomethylation generally occurs in centromeric repeats and repetitive sequences and contributes to carcinogenesis by causing chromosomal instability, reactivation of transposable elements, and loss of imprinting . Hypermethylation is especially frequent in CpG islands, i.e. short DNA sequences rich in CpG dinucleotides, mostly located in the 5'-untranslated region (5'-UTR) of genes . When CpG islands are heavily methylated, transcriptional gene silencing generally occurs. Although the fine mechanisms of regulation of the "epigenetic" machinery are still poorly understood, the DNA methylation may switch on or off several genes and, in particular, those regulating important biological phenomena, such as cell growth and differentiation . In normal cells, this epigenetic mechanism is involved in several physiological events, such as the inactivation of X chromosome in female cells, silencing either paternal or maternal alleles of "imprinted" genes, and transcriptional blocking of exogenous integrated genes potentially dangerous for the cell life. However, aberrant DNA methylation is also relatively common in cancer cells and is likely to play an important role in cancer initiation and progression .
Since the pioneer studies of Baylin et al. , it has been widely recognized that cancer cells are characterised by two opposite events: a global hypomethylation which results in either up-regulation of proto-oncogenes and induction of genomic instability, favouring both uncontrolled cell growth  and mutations, and CpG islands hypermethylation of other genes, the so-called tumour-suppressor-genes (TSG), which contributes to loss of the negative control of the cell cycle . Searching for up-regulated oncogenes and down-regulated TSG is important in basic science, especially when an epigenetic mechanism (hypomethylation or hypermethylation) is suspected. In fact, oncogenes and TSG not only may elucidate the highly complex molecular derangement in cancer cells, but also may be used as potential targets for new therapeutic approaches. DNA methylation is a reversible phenomenon which can be modulated by specific agents. An example is represented by demethylating drugs which can globally reduce the DNA methylation level of TSG promoters, restoring their normal activity. Interestingly, some in vitro experiments have shown that cancer cell lines reverted to normal phenotype after treatment with demethylating agent.
The current study was carried out with a three-step design. First, we specifically looked at up- and down-regulated genes not yet firmly associated with colon carcinogenesis, and selected 24 genes for validation with qPCR. A straight correlation between results obtained from qPCR and those from DNA microarray was found, implying that DNA microarray technology is a reliable tool to search for new genes significantly deregulated in cancer . Second, we selected 10 of 21 genes (ABCA8, AQP8, HPGD, PRDX6, SLC26A3, STX12, NDRG2, MXI1, SGK2, and SCNNB1) as possible targets of epigenetic modifications in colon cancer, and after treatment with a demethylating agent, seven of them showed a significant increase of mRNA expression (AQP8, HPGD, PRDX6, MXI1, SCNNB1, SGK2 and NDRG2). From an in silico screening, only 2 genes (PRDX6 and NDRG2) were considered as possible candidates for the presence of CpG islands in their 5'-UTR. For the excluded genes, additional mechanisms of transcriptional regulation were hypothesized to be responsible for their differential expression. Third, to evaluate the methylation status of PRDX6 an NDRG2 genes in normal and cancer tissues, as well as in colon cancer cell lines, bisulphite sequencing analysis was used. In the PDRX6 gene the methylation status was not different from that observed in normal tissue. In the NDRG2 gene a significant methylation status either in colon cancer cell lines and in tumour tissue compared to normal tissue was observed. The underexpression of the PRDX6 protein responsible for the red-ox regulation of the cell, was found to be correlated with loss of function of NKX3.1 gene, known as TSG .
Using these approaches, the NDRG2 gene was selected for further analysis because: (i) it was suppressed in all colon cancer cell lines, (ii) its expression may be up-regulated in all cell lines by 5Aza-CdR treatments, and (iii) it is involved in important biological process such as cell growth , differentiation  and apoptosis . The NDRG2 gene is a new member of the N-myc downstream-regulated gene (NDRG) family, that is located on chromosome 14q11.2 and encodes for a 41 kDa protein. It has been proposed that the NDRG2 gene is a candidate TSG, and its expression is low or undetectable in several primary tumour and tumour cell lines [30, 33, 34]. Liu et al.  revealed that the down-regulation reported in cancer be driven by promoter methylation, mutation, and genomic deletion of the NDRG2 gene. Recently, it has been shown that expression of the NDRG2 protein is modulated by the insulin-stimulated Akt-dependent phosphorylation . Several studies have suggested that the NDRG2 mRNA is down-regulated or undetectable in a number of human primary cancers, such as squamous cell carcinoma, pancreatic cancer , glioblastoma , and cancer cell-lines. Recently, Zhang et al.  have demonstrated that c-Myc represses NDRG2 gene expression via Miz-1-dependent interaction with NDRG2 core promoter region, and this inverse regulatory relationship induces cell differentiation and proliferation.
The MSP assay was used to check for NDRG2 methylation status in 30 primary colon tumour tissues compared to normal colonic mucosal samples. After sorting colon cancer patients by age, gender, tumour site, and MSI status, no statistically significant association was observed between these features and the NDRG2 methylation. Nevertheless, there was a trend towards NDRG2 methylation status with an advanced tumour stage of the CRC samples, with significant value detected in patients with AJCC stage IV (p < 0.05). These results are in agreement with those reported in other cancer types [34, 39] where NDRG2 expression is reduced in high-grade compared to low-grade tumours. In particular, Lorentzen et al.  suggested that in CRC samples the down-regulation of NDRG2 expression occurs during the progression from adenoma to carcinoma.