Application of LC-MS/MS to the searching of methylated exons in colorectal cancer tissues

Tumor suppressor genes (TSGs) with DNA methylation has been suggested as effective biomarkers for cancer identification and promoter methylation in TSGs has been widely discussed. However, the feasibility of TSG exon methylation used for discriminating cancers was not fully clarified yet. In this study, genomic DNA was isolated from colorectal tissues and responding cancer-adjacent counterparts, and then bisulfite-converted. The exon 1 region of four popular TSGs including ALX4, FBN1, MLH1 and BCL2 was amplified from bisulfite-treated DNA, using nest- polymerase chain reaction (PCR) technique. The purified amplicon was hydrolyzed and then used to detect the methylation level using a robust and convenient LC-MS/MS method. The methodological validation revealed the favorable sensitivity and accuracy of established LC-MS/MS approach. The LC-MS/MS result showed that the methylation level of ALX4 and FBN1 exon in colorectal cancerous tissues (23.70 ± 10.85 and 33.23 ± 6.64) was significant higher than that in cancer-adjacent tissues (12.15 ± 7.08 and 22.08 ± 4.46) with statistic difference, and their value of area under curve (AUC) in receiver operator characteristic curve (ROC) analysis were higher than 0.8. On the other hand, the methylation level of MLH1 and BCL2 was 13.95 ± 6.93 and 15.46 ± 2.41 in CRC tissues and 13.87 ± 3.47 and 12.84 ± 1.91 in cancer-adjacent tissues, respectively. It is demonstrated that the methylation alteration of MLH1 and BCL2 exhibited a non-differential association between two groups. The finding indicated exon methylation of ALX4 and FBN1 could effectively distinguish CRC from non-CRC tissue and exon-based methylation should be used as potential biomarkers for cancer identification.