Differential Gene Expression and Tumorigenicity Analysis of Cultured Melanocyte Comparing Melanoma.

Abstract

This study aimed to identify the optimal growth media for culturing human skin melanocytes for clinical applications and to assess their tumorigenic potential both in vitro and in vivo. Various growth media were tested to determine the most effective and safest for melanocyte culture, avoiding harmful growth factors such as TPA and colorant toxins. The study evaluated changes in RAF and NRAS gene expression through real-time PCR and gene sequencing of BRAF V600E and NRAS in exons 1 and 2, comparing these with melanoma. Melanocytes were subcutaneously injected into BALB/c nude mice to assess tumorigenic risk. Results indicated that a mixture of MGM-M2 supplemented with melanocyte growth factors provided the best outcomes in terms of cell proliferation and melanocyte count. Gene expression analysis revealed that HRAS and BRAF expressions in melanocytes at passage 6 showed less than 2-fold increases, whereas these genes were up-regulated by more than 3 and 8 folds, respectively, in melanoma cell lines. NRAS expression in melanocytes at passage 6 increased by 5-fold but remained lower than in melanoma cell lines. Gene sequencing of BRAF V600E and NRAS in exons 1 and 2 showed no mutations, and melanocytes injected into BALB/c nude mice exhibited no tumor formation risk. Furthermore, gene sequencing of BRAF and NRAS in the injected melanocytes 16 weeks' post-transplantation revealed no mutations. These findings suggest that while standard growth media protocols may elevate specific proto-oncogene expressions, they do not induce tumorigenic mutations in melanocytes, both in vitro and in vivo.