Identification of novel hypermethylated genes and demethylating effect of vincristine in colorectal cancer

Abstract

Background: Colorectal cancer (CRC) arises as a consequence of genetic events such as gene mutation and epigenetic alteration. The aim of this study was to identify new hypermethylated candidate genes and methylation-based therapeutic targets using vincristine in CRC.

Methods: We analyzed the methylation status of 27,578 CpG sites spanning more than 14,000 genes in CRC tissues compared with adjacent normal tissues and normal colon tissues using Illumina bead chip array. Twenty-one hypermethylated genes and 18 CpG island methylator phenotype markers were selected as candidate genes. The methylation status of 39 genes was validated by quantitative methylation-specific polymerase chain reaction in CRC tissues, adjacent normal tissues, normal colon cells, and three CRC cell lines. Of these, 29 hypermethylated candidate genes were investigated using the demethylating effects of 5-aza-2'-deoxycytidine (5-aza-dC) and vincristine in CRC cells.

Results: Thirty-two out of 39 genes were hypermethylated in CRC tissues compared with adjacent normal tissues. Vincristine induced demethylation of methylated genes in CRC cells to the same extent as 5-aza-dC. The mRNA expression of AKR1B1, CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304 was restored by treatment with 5-aza-dC and vincristine.

Conclusion: These results suggest that these novel hypermethylated genes AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 may be potential methylation biomarkers and therapeutic targets of vincristine in CRC.

Figure 1

Hypermethylation of promoter CpG sites for 21 genes by methylation chip array. The methylation statuses of CpG sites in promoter CpG islands for 21 genes were identified using methylation chip array in 10 normal colon tissues, 21 CRC tissues, and 21 adjacent normal tissues. All genes were significantly strongly hypermethylated in their promoter CpG islands in CRC tissues compared with adjacent normal tissues and normal tissues (P < 0.05). N: normal colon tissues, T: CRC tissues, AN: adjacent normal tissues.

Figure 2

Validation of methylation status of 21 candidate genes and 18 CIMP markers. The methylation status of 21 candidate genes and 18 CIMP markers was validated using QMSP in 10 CRC tissues compared with adjacent normal tissues (A), normal colon cells, and three CRC cell lines (B). Methylated intensity ratio of QMSP data was determined by the PMR values. The significance of different methylation statuses in CRC tissues and adjacent normal tissues was defined with the analysis of variance test using Sigma Stat. Values of P < 0.05 were considered to indicate statistical significance. * indicates the methylation status of genes in CRC compared with adjacent normal tissues was not statistically significant.

Figure 3

Demethylating effect of 5-aza-dC and vincristine on methylated genes in three CRC cell lines. Changes in the methylation status of hypermethylated genes in normal colon cells (A) and CRC cells (B) were identified by QMSP after treatment with 30 μM 5-aza-dC or 100 nM vincristine for 48 h at 37°C. Methylated intensity ratio was determined by the percentage of methylated reference (PMR).

Figure 4

Association between DNA methylation and mRNA expression of eight genes in CCD18Co and DLD-1. The DNA methylation status (A) and mRNA expression (B) of eight genes, including six newly identified hypermethylated genes, was measured using QMSP and RT-PCR in CCD18Co and DLD-1 cells following 30 μM 5-aza-dC or 100 nM vincristine treatment for 48 h at 37°C. ACTB was amplified simultaneously with the other PCR products and was used as a control for DNA methylation and mRNA integrity. Marker: molecular weight DNA marker.