The Research Of Aberrant Methylation In The Promoter CpG Island Of IGFBP7 Gene In Cutaneous Malignant Melanoma Cells

Cutaneous malignant melanoma is the most aggressive and deadly form of skin cancer. Furthermore,the mortality is still increasing. It is of great significance to study the underlying mechanisms of malignant melanoma for its prevention and treatment. IGFBP7 is one of the most important tumor suppressor genes in malignant melanoma. Many studies suggest that hypermethylation of the 5'-flanking promoter region CpG islands silences tumor suppressor genes. In this research, we investigated the relation about abnormal DNA methylation of promoter region CpG islands and the gene expression of IGFBP7.1 The expression of IGFBP7 in cutaneous melanoma tissues and cell linesImmunohistochemical examination revealed that the level of IGFBP7 was significantly lower in melanoma than in benign pigmented nevi tissues. Semi-quantitative reverse transcription-PCR(RT-PCR) and immunohistochemical examination were applied to evaluate the expression of IGFBP7 in 4 melanoma cell lines(A375,M14,SK-MEL-1,MV3) and original melanocytes. IGFBP7 was only expressioned in MV3 cell and original melanocytes.2 The analysis of the methylation status of the promoter region of IGFBP7 in melanoma cell linesBisulfite sequencing PCR(BSP) was applied to detect the methylation status of 54 CpG sites in the 5'-flanking promoter region CpG island of IGFBP7 gene in all of the 4 melanoma cell lines and original melanocytes. Hierarchical cluster analysis showed the methylation status of the promoter region was significantly different between IGFBP7-positive and-negative cells.3 The effect of demethylation agent 5-aza-dC on the IGFBP7 expression and the biological processes of melanoma cell linesIn order to further study whether DNA methylation is the direct cause of aberrant IGFBP7,2 melanoma cell lines A375, M14 were treated with demethylation agent 5-aza-dC. Results showed that 5-aza-dC induced demethylation of IGFBP7 gene and restored IGFBP7 expression both at mRNA and protein levels. It suggest that upregulation of IGFBP7 expression was the direct effect of demethylation.We also found the inhibited cell proliferation, arrested cell cycle, increased cell apoptosis and decreased migration ability of melanoma cells after treatment with 5-aza-dC. Thus we proposed that restoration of IGFBP7 expression might be involved in these biological processes.Based on the results above, we drew the following conclusions:IGFBP7 gene promoter region CpG island DNA methylation is the main regulatory mechanism underlyning aberrant IGFBP7 expression in melanoma cell lines. Demethylation agent 5-aza-dC can induce hypomethylation and reexpression of IGFBP7. Restoration of IGFBP7 expression may participate in the processes of the inhibition of cell growth, arrest of cell cycle, elevation of cell apoptosis and reduction of migration of melanoma cell lines.