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Abundant mucin production and MUC2 expression is the key feature of mucinous colorectal cancer (CRC). Although MUC2 gene methylation has been thought to play an important role in loss of MUC2 expression, the tissues are difficult to analyze because of the cellular heterogeneity of tissue samples. In the present study, we determined the role of region-specific methylation in the MUC2 promoter in MUC2 expression in CRC. Additionally, we optimized the conditions for quantification of methylation analysis in mucinous and non-mucinous CRC tissues. We identified two regions in MUC2 promoter, region A (-289 and -274) and region C (-193 and -160), that correlated with loss of MUC2 expression by comparing the methylation status in 13 CRC cell lines with no or low MUC2 expression and those in 4 cell lines with high MUC2 expression. To prove the correlation of MUC2 methylation status and loss of expression in CRC tissues, MUC2 methylation status in tumors needs to be determined. Since the critical CpG sites have been identified in cell lines by sequencing, a more rapid and sensitive methylation specific PCR (MSP) was used. We conducted MSP at 3 CpG sites (-289, -274, -193) in 19 mucinous and 34 non-mucinous CRC tissues because this analysis worked at only these sites in the preliminary cell line experiments. Our results showed that methylation status of mucinous CRC was significantly lower than that of non-mucinous CRC at 3 sites (-289; p=0.001, -274; p=0.013, -193; p=0.001), and correlated with high level of MUC2 expression as determined by immunohistochemistry. Besides, these results indicated that MUC2 expression and mucin contents decreased in accordance with the increase of methylation status. We concluded that low methylation status of MUC2 gene plays a predominant role in high level MUC2 expression in mucinous CRC.

MUC2 gene promoter methylation in mucinous and non-mucinous colorectal cancer tissues