Preliminary Exploration of MAGE-B1, -B4, -B5, and -B10 mRNA Expression in Canine Mammary Tumors in Dogs

Abstract

The melanoma-associated antigen gene (MAGE) is a key target in cancer immunotherapy. Given the potential of MAGE-B genes in veterinary immunotherapy for canine mammary tumors (CMTs), this study investigated the mRNA expression of MAGE-B1, -B4, -B5, and -B10 in CMT tissues and cells from dogs. Quantitative real-time PCR was used to analyze 28 CMT tissue samples, including 4 benign and 24 malignant tumors (13 simple carcinomas, 6 complex carcinomas, 3 carcinosarcomas, and 2 fibrosarcomas). Benign mixed tumor and complex carcinoma-type CMT cells were cultured and treated with a DNA methylase inhibitor (5-aza-2'-deoxycytidine; 5-aza-CdR) and a histone deacetylase inhibitor (Trichostatin A; TSA) under the following four conditions: (1) 5-aza-CdR for 72 h; (2) TSA for 24 h; (3) 5-aza-CdR for 48 h followed by TSA for 24 h; and (4) control. MAGE-B1 and -B4 showed the highest expression in the CMT samples (100% and 89.29%, respectively), followed by MAGE-B10 (82.14%). Carcinosarcomas and simple anaplastic carcinomas had significantly higher MAGE-B expression levels than simple tubulopapillary carcinomas (p < 0.05). 5-aza-CdR treatment increased MAGE-B expression, whereas TSA had a mild effect. Further research involving larger cohorts is needed to confirm these findings.

Keywords: canine mammary tumors; mRNA expression; melanoma-associated antigen-B; quantitative real-time PCR.

Figure 1

The relative fold change in the mRNA expression of MAGE-B1, -B4, -B5, and -B10 from canine mammary tumor tissues using GAPDH as a reference gene [46]. The heat map presents the fold change in 2^{(−∆∆CT)} values. The lack of data means there was no mRNA expression detected (Ct cycle > 35). The fold change expression ranged between 0 and 0.20. The 0 value or green color means the level of expression is low, while the 0.20 value or red color means the level of expression is high.

Figure 2

The comparisons of mean level of relative fold change in the mRNA expression results of MAGE-B1, -B4, -B5, and -B10 genes between benign and malignant CMTs. (a): A comparison of the mean level of relative fold change in the mRNA expression results of MAGE-B1 between benign CMTs (n = 4) and malignant CMTs (n = 24). (b): A comparison of the mean level of relative fold change in the mRNA expression results of MAGE-B4 between benign CMTs (n = 4) and malignant CMTs (n = 24). (c): A comparison of the mean level of relative fold change in the mRNA expression results of MAGE-B5 between benign CMTs (n = 4) and malignant CMTs (n = 19). (d): A comparison of the mean level of relative fold change in the mRNA expression results of MAGE-B10 between benign CMTs (n = 3) and malignant CMTs (n = 20).

Figure 3

The comparison of the mean relative fold change in the mRNA expression results of MAGE-B genes between different histopathological types of canine mammary gland tumor tissue samples using the nested t-test analysis [46]. * Different significant values between groups were at p ≤ 0.01 and ** different significant values between groups were at p < 0.05.

Figure 4

The effects of 5-aza-CdR and/or TSA on the relative fold change in MAGE-B1, -B4, -B5, and -B10 mRNA expression in the benign type of CMT primary cells. (a): The relative fold change in MAGE-B1 mRNA expression in the benign type of CMT primary cells in different treatments. (b): The relative fold change in MAGE-B4 mRNA expression in the benign type of CMT primary cells in different treatments. (c): The relative fold change in MAGE-B5 mRNA expression in the benign type of CMT primary cells in different treatments. (d): The relative fold change in MAGE-B10 mRNA expression in the benign type of CMT primary cells in different treatments.

Figure 5

The effects of 5-aza-CdR and/or TSA on the relative fold change in MAGE-B1, -B4, -B5, and -B10 mRNA expression in the malignant type of CMT primary cells. (a): The relative fold change in MAGE-B1 mRNA expression in the benign type of CMT primary cells in different treatments. (b): The relative fold change in MAGE-B4 mRNA expression in the benign type of CMT primary cells in different treatments. (c): The relative fold change in MAGE-B5 mRNA expression in the benign type of CMT primary cells in different treatments. (d): The relative fold change in MAGE-B10 mRNA expression in the benign type of CMT primary cells in different treatments.