ABSTRACT
Identification of methylation-silenced genes in colorectal cancer (CRC) is of great importance. We employed oligonucleotide microarrays to identify differences in global gene expression of five CRC cell lines (HCT116, RKO, Colo320, SW480 and HT29) that were analyzed before and after treatment with 5-aza-2'-deoxycitidine. Selected candidates were subjected to methylation-specific PCR and real-time quantitative reverse transcription-PCR using 15 CRC cell lines and 23 paired tumor and normal samples from CRC patients. After 5-aza-2'-deoxycitidine treatment, 139 genes were re-expressed in all 5 CRC cell lines collectively with a fold change of more than 1.5 in at least one cell line. These genes include known methylated and silenced genes in CRC. After applying study selection criteria we identified 20 candidates. The GADD45B and THSD1 genes were selected for further analysis. Among 15 colon cancer cell lines, methylation was only identified in THSD1 (27%). THSD1 methylation was subsequently investigated in 23 colorectal tumors and methylation was detected in 9% of the analyzed samples; the observed promoter hypermethylation was cancer-specific. THSD1 mRNA down-regulation was observed in tumor tissues. This genome-wide screening led to the identification of genes putatively affected by methylation in CRC. The THSD1 gene may play a role in the tumorigenesis of CRC.
Subject(s)
Antigens, Differentiation/genetics , Colorectal Neoplasms/genetics , Gene Silencing , Thrombospondins/genetics , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , DNA Methylation , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genome-Wide Association Study , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Promoter Regions, GeneticABSTRACT
AIM: Unearthing of silenced genes in colorectal cancer (CRC). MATERIALS AND METHODS: Oligonucleotide microarray was used in order to find changes in gene expression in five CRC cell lines before and after 5-aza-2'-Deoxycitidine treatment. Up-regulated genes were integrated with expression profile of matched colorectal tissue samples. Methylation-specific polymerase chain reaction and Real-time quantitative reverse transcription polymerase chain reaction were used to further analyze candidates using 15 CRC cell lines and 23 paired samples. RESULTS: After applying study selection criteria for 68 genes obtained from integrated arrays, we identified 16 genes; apoptosis-stimulating of p53 protein 1(ASPP1) and Scavenger receptor class A, member 5 (SCARA5) were selected for further analysis. Methylation was only identified for SCARA5 in 20% of the cell lines and in 17% of tumor the samples. Down expression of SCARA5 was observed in CRC cell lines and in tumor samples compared to normal (p<0.001 and p=0.001, respectively). CONCLUSION: Genome-wide screening identifies genes potentially affected by methylation in CRC. SCARA5 may have a role in tumorigenesis in CRC.
