Investigation of the function of genes expressed in melanoma/melanocyte with RNA interference for understanding of pigment disorders

我们试图通过干扰抑制基因表达来分析黑素细胞特异性基因(MART-1和AIM-1)和高表达基因(β-连环蛋白和FABP7)在黑色素瘤中的功能。首先，我们构建了一个测量系统，可以通过黑色素瘤细胞中的黑色素含量来推断RNA干扰的效率。然后，我们试图通过每个特定的siRNA下调MART-1和AIM-1的表达。MART-1和AIM-1的表达分别为对照细胞的50%和30%。但黑色素瘤细胞的表型没有变化，至少在生长和迁移方面没有变化。β-Catenin是黑色素瘤细胞中的一种异常积聚蛋白，β-Catenin特异的小干扰RNA可下调其表达。624AMEL和888meL细胞经特异性siRNA处理后，β-catenin蛋白表达水平下降不到对照细胞的20%，细胞增殖率(Wst-1法)降低至正常细胞的70%~80%。然后用DNA芯片鉴定FABP7基因在黑色素瘤细胞系中高表达。当FABP7特异性siRNA下调FABP7的表达时，黑色素瘤细胞株WM266mel和888mel的体外细胞增殖和Matrigel迁移均受到抑制。反之，携带FABP7的293T细胞的增殖和Matrigel迁移能力增强。这些结果提示FABP7可能参与了黑色素瘤恶性表型的形成。未检测到MART-1和AIM-1的黑色素含量或其他表型在RNA干扰细胞和对照细胞之间的差异。在含有β-catenin和FABP7特异性siRNA的黑色素瘤细胞中，我们可以观察到细胞增殖受到抑制，我们也可以检测到FABP7对细胞迁移的抑制。我们认为RNA干扰是发展功能分析和分子靶向治疗的一个非常有用的工具。

We attempted to analyze function of melanocyte-specific genes(MART-1 and AIM-1) and highly expressed genes (β-catenin and FABP7) in melanoma by suppression of gene expression using RNA interference. First we constructed a measurement system that could deduce efficiency of RNA interference by the content of melanin in pigmented melanoma cells. Then we tried to downregulate the MART-1 and the AIM-1 expression with each specific siRNA. The expression of MART-1 and AIM-1 was lowered to 50 and 30 %, respectively, of that in control cells. But there were no changes in the phenotype of melanoma cells at least in growth and migration. β-catenin known as an aberrant accumulated protein in melanoma cells was downregulated with β-catenin specific siRNA. β-catenin protein of 624Amel and 888mel with specific siRNA was decreased less than 20% of control cells and cell proliferation(WST-1 assay) of 624Amel and 888mel with specific siRNA was decreased to 70-80% compared to normal cells. FABP7 identified as a highly expressed gene in melanoma cell lines by DNA chips was then used. When the FABP7 expression was downregulated with FABP7 specific siRNA, in vitro cell proliferation and Matrigel migration of melanoma cell lines, WM266mel and 888mel, were decreased. In contrary, cell proliferation and Matrigel migration of 293T cells with plasmid FABP7 was increased. These results suggest that FABP7 may be involved in formation of malignant phenotype of melanoma. We could not detect deference of melanin content or other phenotypes between RNA interfered cells and control cells about MART-1 and AIM-1. In melanoma cells with β-catenin and FABP7 specific siRNA, we could observe suppression of cell proliferation, and we can also detect suppression of cell migration about FABP7. We think RNA interference is a very useful tool for development of functional analysis and molecular target therapy.