117 Methylation Regulation of Laminin Alpha 5 Gene in Human Melanoma Progression

Recently, gene methylation has come to the forefront of melanoma research, highlighting unique perspectives on the gene regulation in melanoma progression and metastatic potential. Laminin alpha 5 is a major extracellular component of basement membrane of the epithelia and blood vessels and has been shown increased expression in some malignant tumors. The purpose of this study is to evaluate the potential role of methylation of laminin alpha 5 gene in melanoma cell malignant behavior. Normal skin melanocytes, Sk-Mel-28 (skin primary melanoma) and Mewo (lymph node metastatic melanoma) cell lines were used in this study. Genomic DNAs were extracted and purified with NucleoSpin® Tissue Kit. Bisulfate conversion was used to detect methylation of laminin α5 (EZ DNA methylation kit). This technique converts unmethylated cytosines into uracil. Methylated cytosines remain unchanged during the treatment. PCR amplifications were performed using specially designed laminin alpha 5 primers (Wild type, Methylated and Unmethylated) and OneTaq® Hot Start DNA Polymerase. Methylated primers were used to evaluate the level of DNA methylation. The DNA bands were separated with 1% agarose gel and visualized by Ethidium Bromide staining and UV imaging system. The results showed that Sk-mel-28 cells DNA had much high level of laminin alpha 5 methylation compared to Mewo cells. No significant bands were seen with unmethylated primers. The results suggests that methylation of laminin alpha 5 gene may be involved in melanoma progression and metastasis.