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

我们试图通过RNA干扰抑制黑色素细胞特异性基因（MART-1和AIM-1）和高表达基因（β-catenin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定的siRNA下调MART-1和AIM-1的表达。MART-1和AIM-1的表达分别降低到对照细胞的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累蛋白，它被β-catenin特异性siRNA下调。与正常细胞相比，624Amel和888mel的β-catenin蛋白下降幅度小于对照细胞的20%，624Amel和888mel的细胞增殖（WST-1试验）下降幅度为70-80%。然后使用DNA芯片鉴定出的高表达基因FABP7在黑色素瘤细胞系中。当用FABP7特异性siRNA下调FABP7的表达时，黑色素瘤细胞系WM266mel和888mel的体外细胞增殖和基质迁移均下降。相反，携带质粒FABP7的293T细胞增殖能力和基质迁移能力增强。这些结果提示FABP7可能参与黑色素瘤恶性表型的形成。我们没有检测到RNA干扰细胞和对照细胞在MART-1和AIM-1的黑色素含量或其他表型上的差异。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，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.