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Screening for Epigenetically Masked Genes in Colorectal Cancer using 5-aza-2’-deoxycytidine treatment, Microarray and Gene Expression Profile

ABSTRACT: Unearthing of silenced genes in colorectal cancer (CRC) is of great importance. We employed oligonucleotide microarray to find changes in global gene expression of five CRC cell lines. These were analyzed before and after treatment with the 5-aza-2'-Deoxycitidine. Expression of the responding genes was integrated with gene expression profiling generated by microarray analysis of matched colorectal tissue samples. Selected candidates were subjected to methylation-specific PCR (MSP) and real-time quantitative reverse transcription-PCR using CRC cell lines and paired tumor and normal samples from CRC patients. Sixty eight genes were re-expressed after 5-aza-2'-Deoxycitidine treatment and over-expressed in normal colorectal mucosa, including genes that were known to be methylated in CRC. After applying study selection criteria, we identified 16 potential genes. Two candidates were selected (ASPP1 and SCARA5). Among 15 CRC cell lines, methylation was identified in SCARA5 (20%). The methylation status of SCARA5 was subsequently investigated in 23 paired colorectal tissue samples; methylation was detected in 17%, respectively. Observed promoter methylation showed a tendency towards methylation in tumor-derived samples, in SCARA5 gene. Significant down expression of SCARA5 mRNA was observed in CRC cell lines and tumor tissues compared to adjacent normal tissues (P < 0.001 and P = 0.001, respectively). The use of genome-wide screening led to the identification of a group of candidate genes. Among them, SCARA5 was methylated and markedly down-regulated in CRC. SCARA5 gene may have a role in CRC tumorigenesis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE32323 | GEO | 2012/03/20

SECONDARY ACCESSION(S): PRJNA147273

REPOSITORIES: GEO

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Project description:Colorectal cancer (CRC) is a genetically heterogeneous disease with several distinct morphological growth patterns. This study was aimed to investigate genes differentially expressed between ulcerative and polypoid colorectal CRC. cDNA microarray was first employed to compare the gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa. Potential candidates identified by data filtering were further validated using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Epigenetic regulation of gene expression was investigated using methylation-specific PCR (MSP). cDNA microarray identified 11 up-regulated and 14 down-regulated genes differentially expressed in both types of tumor compared to matched normal mucosa. Among them, S100P was the only upregulated gene preferentially associated with polypoid CRC (p = 0.032), whereas no genes demonstrated significantly differential association with ulcerative CRC. S100P protein and mRNA expression level of polypoid CRC was significantly higher than that of ulcerative CRC (p < 0.05, respectively). Immunoreactivity of S100P protein was localized predominantly in the nuclei and to a less extent in the cytoplasm. Overexpression of S100P occurred early in the adenoma stage. 80% (24/30) and 20% (6/30) polypoid CRC showed diffusely strong and moderate overexpression, respectively. In contrast, S100P was diffusely and strongly expressed in 15% (6/40) ulcerative CRC, with 52.5% (21/40) and 32.5% (13/40) tumors having moderate and weak overexpression, respectively. S100P overexpression was preferentially associated with polypoid CRC (p < 0.001). The relative methylation level determined by MSP was not statistically different between polypoid and ulcerative CRC (43.36% vs. 49.10%, p = 0.168), indicating that promoter hypomethylation was directly related to upregulation of S100P mRNA. The gene expression profiling of ulcerative and polypoid CRC with paired normal mucosa were compared. Dye swapping experiments with ulcerative CRC vs normal mucosa or polypoid CRC vs normal mucosa were performed and we averaged the log ratios of the duplicated spots on each slide.

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Screening for Epigenetically Masked Genes in Colorectal Cancer using 5-aza-2’-deoxycytidine treatment, Microarray and Gene Expression Profile

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