Abstract
BackgroundColorectal cancer (CRC) is one of the leading causes of death by cancer worldwide and in need of novel potential diagnostic biomarkers for early discovery.MethodsWe conducted a two-step study. We first employed bioinformatics on data from The Cancer Genome Atlas to obtain potential biomarkers and then experimentally validated some of them on our clinical samples. Our aim was to find a methylation alteration common to all clusters, with the potential of becoming a diagnostic biomarker in CRC.ResultsUnsupervised clustering of methylation data resulted in four clusters, none of which had a known common genetic or epigenetic event, such as mutations or methylation. The intersect among clusters and regulatory regions resulted in 590 aberrantly methylated probes, belonging to 198 differentially expressed genes. After performing pathway and functional analysis on differentially expressed genes, we selected six genes: CEP55, FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5, for further experimental validation on our own clinical samples. In silico analysis demonstrated that CEP55 was hypomethylated in 98.7% and up-regulated in 95.0% of samples. Genes FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 97.9, 81.1, 80.3, 98.4 and 94.0%, and down-regulated in 98.3, 98.9, 98.1, 98.1 and 98.6% of samples, respectively. Our experimental data show CEP55 was hypomethylated in 97.3% of samples and down-regulated in all samples, while FOXD3, FOXF2, GNAO1, GRIA4 and KCNA5 were hypermethylated in 100.0, 90.2, 100.0, 99.1 and 100.0%, and down-regulated in 68.0, 76.0, 96.0, 95.2 and 84.0% of samples, respectively. Results of in silico and our experimental analyses showed that more than 97% of samples had at least four methylation markers altered.ConclusionsUsing bioinformatics followed by experimental validation, we identified a set of six genes that were differentially expressed in CRC compared to normal mucosa and whose expression seems to be methylation dependent. Moreover, all of these six genes were common in all methylation clusters and mutation statuses of CRC and as such are believed to be an early event in human CRC carcinogenesis and to represent potential CRC biomarkers.
Highlights
Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide and in need of novel potential diagnostic biomarkers for early discovery
Bioinformatics analysis was performed on samples from projects Colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) obtained from The Cancer Genome Atlas (TCGA)
Methylation analysis on the same cohort of samples (n = 25) revealed that, in CRC compared to normal mucosa, gene CEP55 was completely hypomethylated and up-regulated
Summary
Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide and in need of novel potential diagnostic biomarkers for early discovery. CRC occurs through a process of malignant transformation, when numerous genetic and epigenetic events transform normal colon mucosa to adenocarcinoma [2]. It is a very heterogeneous disease, in which three major molecular pathways have been identified. The microsatellite instability (MSI) pathway accounts for approximately 15% of sporadic CRC, and is characterized by deficiency in DNA mismatch repair (MMR) genes (e.g. MLH1, MSH2, MSH6, PMS2) [4]. Silencing of MMR genes in the MSI type of CRC occurs through promoter hypermethylation, a common molecular alteration at epigenetic level. The third molecular pathway is the CpG island methylator phenotype (CIMP); an epigenetic instability pathway. One of these three pathways is usually predominant but they are not mutually exclusive [6, 7]
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