hMAGEA2 is highly expressed in human melanoma tissues
hMAGEA2 is overexpressed in a variety of human cancers, and its overexpression was found to be correlated with a worse prognosis [5, 10-13]. To investigate whether hMAGEA2 is overexpressed in human melanoma tissues and whether MAGE expression increases with metastasis, we measured expression levels of hMAGEA2 in human melanoma tissues using TMAs. Results showed that the expression level of hMAGEA2 was significantly upregulated in human melanoma cancer tissues compared with adjacent cancer tissues and normal tissues (P < 0.01) (Figure 1A, 1B), and hMAGEA2 expression was significantly higher in melanoma with metastasis than in melanoma without metastasis (P < 0.01) (Figure 1C). These findings suggest that hMAGEA2 is associated with melanoma metastasis and tumorigenesis.
Overexpression of hMAGEA2 in melanoma cell lines promotes cell growth.
To investigate the functional relevance of hMAGEA2 in melanoma, we established SK-MEL-5 and SK-MEL-28 cell lines that overexpress hMAGEA2 by stable transfection using a plasmid vector not expressing hMAGEA2 (mock vector) or a vector that drives the expression of hMAGEA2. Immunoblot analysis results showed that the expression of hMAGEA2 protein was increased significantly in SK-MEL-5 and SK-MEL-28 cell lines compared with mock-transfected cell lines (Figure2A). Cell proliferation was measured by the CCK-8 proliferation assay. Proliferation was measured at 0, 24, 48, 72, and 96h in the stably transfected SK-MEL-5 and SK-MEL-28 cell lines. The results showed that the proliferation was increased in cell lines that overexpress hMAGEA2 relative to control cells (Figure2B). Our results demonstrate that the overexpression of hMAGEA2 in SK-MEL-5 and SK-MEL-28 increases cell proliferation.
Knockdown of hMAGEA2 in melanoma cells suppresses cell growth
To investigate the effects of hMAGEA2 downregulation, we established cells in which the overexpression of hMAGEA2 was suppressed in the stably transfected SK-MEL-5 and SK-MEL-28 cell lines using lentivirus infection. Immunoblot analysis of sh-hMAGEA2-, and sh-MOCK-infected melanoma cells revealed that expression of endogenous hMAGEA2 was suppressed relative to cells expressing sh-MOCK (Figure 3A). The results of the CCK-8 proliferation assays indicate that knockdown of hMAGEA2 significantly decreases the proliferation of SK-MEL-5 and SK-MEL-28 cells (Figure 3B). Overall, this data demonstrates that hMAGEA2 protein plays an important role in melanoma cell proliferation.
hMAGEA2 inhibits the migration of melanoma cells
Clinical pathology data analysis indicates that hMAGEA2 is involved in tumor metastasis. To confirm whether hMAGEA2 plays a role in the migration of SK-MEL-5 and SK-MEL-28cells, a wound-healing assay was performed. First, we constructed hMAGEA2 knockdown SK-MEL-5 and 28 cells, and then measured the serum-induced migration of metastatic SK-MEL-5 and 28 cells. sh-hMAGEA2 decreased serum-induced cancer cell migration as measured by wound-healing assays (Figure 4A). Compared to SK-MEL-5 and 28 sh-MOCK cells, SK-MEL-5 and 28 sh-hMAGEA2 cell migration is significantly increased (Figure 4B). These results suggest that hMAGEA2 plays a key role in melanoma metastasis.
hMAGEA2 is involved in the cell cycle of melanoma cells
The effect of hMAGEA2 on cell cycle progression was analyzed by flow cytometry. The results suggest that overexpression hMAGEA2 induces G1 phase distribution arrest in SK-MEL-28 cells (Figure 5A) and that the knockdown of hMAGEA2 decreases G1 S phase in SK-MEL-5 cells (Figure 5B). These findings indicate that hMAGEA2 is associated with cell cycle characteristics.
Knockdown of hMAGEA2 suppresses colony formation of melanoma cells in soft-agar
The effect of suppressing hMAGEA2 expression on the ability of melanoma cells to form colonies in soft-agar was measured. Colony formation is decreased in sh-hMAGEA2 melanoma cells relative to sh-MOCK control cells (Figure 6A). In SK-MEL-5 and SK-MEL-28 cell lines, the number of colonies formed is significantly decreased by knockdown of hMAGEA2 (Figure 6B). Our results demonstrate that the knockdown of hMAGEA2 suppresses colony formation in soft-agar. Overall, the data demonstrate that the hMAGEA2 protein plays a key role in the anchorage independent growth of melanoma cells.
hMAGEA2 enhances xenograft tumor formation
To investigate the growth rate in vivo, we performed a xenograft tumor formation assay. The volume and diameter of tumors derived from SK-MEL-5 cell lines that overexpress hMAGEA2 were significantly increased relative to cells that do not overexpress hMAGEA2 (Figure 7).