Expression Profile of MAGE-B1 Gene and Its Hypomethylation Activation in Colon Cancer

Abstract

Cancer-testis (CT) genes are typically expressed in the testes; however, they have been linked to aberrant expression in a variety of malignancies. MAGE-B family genes are an example of CT genes. Therefore, the overarching objective of this study was to examine the expressions of MAGE-B family genes in several patients with colon cancer (CC) to see if they might be employed as cancer biomarkers in the early phases of cancer detection and to improve treatment. In this investigation, RT-PCR was used to analyze MAGE-B family genes in neighboring normal colon (NC) tissue from 10 CC patients. In addition, the effect of DNA demethylation on the expression status of the MAGE-B1 gene was evaluated by RT-PCR in HCT116 and Caco-2 cells and by qRT-PCR for HCT116 only after treating both CC cell lines with varying concentrations of 5-aza-2'-deoxycytidine (1.0, 5.0, and 10.0 μM) for 48 or 72 hours. All MAGE-B family genes except for MAGE-B1 showed weak bands in several samples of NC tissues: MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, and MAGE-B6 genes were observed in 40%, 50%, 40%, 30%, and 60% of the NC samples, respectively. Nonetheless, they had strong bands in multiple samples of CC tissues, with 70%, 90%, 60%, 50%, and 90% of the CC samples, respectively. Interestingly, MAGE-B1 was detected in 60% of CC tissues but not in NC tissues, suggesting that it is a potential biomarker for early CC detection. MAGE-B1 expression was not observed in either untreated or DMSO-treated HCT116 cells after 48 or 72 hours of treatment. However, according to the RT-PCR and qRT-PCR results, the MAGE-B1 gene was overexpressed in the HCT116 cells treated with three different concentrations of 5-aza-2'-deoxycytidine. This shows that demethylation plays a crucial role in MAGE-B1 expression activation.

Figure 1

Expression profile of MAGE-B genes in CC and matched adjacent NC tissues. The MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, and MAGE-B6 genes were examined on 1% agarose gels. Total RNA from 10 NC tissues (left side) and 10 CC tissues (right side) was used to make cDNAs. Testis cDNA was used to validate the primers for each gene. As a positive control for the cDNA samples, ACTB expression was used. The expected size of each gene's product is indicated on the left between brackets.

Figure 2

The effects of 5-aza-2′-deoxycytidine on MAGE-B1 gene expression in the CC cell lines. MAGE-B1 gene expression was exhibited in 1% agarose gels following treatment with a variety of 5-aza-2′-deoxycytidine doses (1.0, 5.0, and 10.0 μM) for 48 h (left side) or 72 hours (right side). Untreated HCT116 and Caco-2 cells were used for comparison with treated cells. Testis cDNA was used to validate the primer efficiency of the MAGE-B1 gene. HCT116 and Caco-2 cells were treated with DMSO as a control, as DMSO was utilized to prepare the 5-aza-2′-deoxycytidine solution. As a positive control for the cDNA samples, ACTB expression was used. The expected size of each gene's product is indicated on the left between brackets.

Figure 3

qRT-PCR analysis of MAGE-B1 expression in the HCT116 cell line. The gene expression data for MAGE-B1 in the HCT116 cell line is shown in the bar chart. The GAPDH reference gene was used to normalize the expression data. The error bars indicate the standard error of the mean for three repeats (^∗P < 0.05 and ^∗∗P < 0.01). N denotes the number of samples.