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

我们试图通过使用RNA干扰抑制基因表达来分析黑色素细胞特异性基因（MART-1和AIM-1）和高表达基因（β-catenin和FABP 7）在黑色素瘤中的功能。首先，我们构建了一个测量系统，可以通过色素性黑色素瘤细胞中黑色素的含量来推断RNA干扰的效率。然后，我们尝试用每种特异性siRNA下调MART-1和AIM-1的表达。MART-1和AIM-1的表达分别降低至对照细胞中的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是黑色素瘤细胞中一种异常积累的蛋白质，用β-catenin特异性siRNA下调β-catenin。与正常细胞相比，具有特异性siRNA的624 Amel和888 mel的β-连环蛋白蛋白质降低小于对照细胞的20%，并且具有特异性siRNA的624 Amel和888 mel的细胞增殖（WST-1测定）降低至70-80%。然后使用通过DNA芯片鉴定为在黑素瘤细胞系中高表达的基因FABP 7。当用FABP 7特异性siRNA下调FABP 7表达时，黑色素瘤细胞系WM 266 mel和888 mel的体外细胞增殖和Matrigel迁移降低。相反，具有质粒FABP 7的293 T细胞的细胞增殖和Matrigel迁移增加。这些结果提示FABP 7可能参与了黑色素瘤恶性表型的形成。在MART-1和AIM-1的RNA干扰细胞和对照细胞之间，我们没有检测到黑色素含量或其他表型的差异。在具有β-连环蛋白和FABP 7特异性siRNA的黑素瘤细胞中，我们可以观察到细胞增殖的抑制，并且我们还可以检测到关于FABP 7的细胞迁移的抑制。我们认为RNA干扰是一种非常有用的工具，为功能分析和分子靶向治疗的发展。

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.