Functional analysis of candidate antigens identified by DNA microarray for highly expression in melanoma

识别在黑色素瘤细胞中优先表达并参与其恶性表型的分子对于理解黑色素瘤生物学和开发新的诊断和治疗方法非常重要。我们利用GeneChip将黑色素瘤细胞系的表达谱与各种正常组织的表达谱进行比较，并通过RT-PCR确认所选基因的实际表达，鉴定出FABP7、MMA1和KU-MEL-3。通过RNA干扰和重组转导分析了这些基因的功能。通过FABP7特异性sirna下调FABP7的表达，抑制黑色素瘤细胞系体外细胞增殖和基质入侵。通过转染FABP7 cDNA，在FABP7阴性的胚胎肾细胞系293T中过表达FABP7，导致细胞增殖和Matrigel侵袭增强，表明FABP7在某些黑色素瘤细胞系的恶性表型中起作用。MMA1特异性sirna下调黑色素瘤细胞系体外细胞增殖和细胞粘附抑制通过转染MMA1重组蛋白，在MMA1低表达的黑色素瘤细胞系中过表达MMA1，导致细胞增殖和粘附增强，表明MMA1也在黑色素瘤细胞的恶性表型中发挥作用。免疫组化检查显示，15例黑色素瘤组织中有11例表达FABP7。25例黑色素瘤患者血清中有14例（56%）检测到噬菌体或细菌重组FABP7蛋白的IgG抗体，31例黑色素瘤患者血清中有8例（26%）检测到噬菌体或细菌重组FABP7蛋白的IgG抗体，但在健康个体血清中未检测到，表明FABP7是黑色素瘤患者的免疫原性抗原。这些结果表明，在黑色素瘤中频繁表达的FABP7和MMA1可能是开发诊断和治疗方法的潜在靶点。

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding of melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip, and by confirming the actual expression of the selected genes by RT-PCR, we identified FABP7,MMA1 and KU-MEL-3. These genes were analyzed for their function by RNA interference and recombinant transduction. By down-regulating the FABP7 expression with FABP7 specific siRNAs, in vitro cell proliferation and Matrigel invasion were suppressed in melanoma cell lines. Over-expression of FABP7 in a FABP7 negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA, resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. MMA1 down-regulation with MMA1 specific siRNAs suppressed in vitro cell proliferation and cell adhesion in melanoma cell lines. Over-expression of MMA1 in the melanoma cell lines with low MMA1 expression by transfecting with the MMA1 recombinant resulted in enhanced cell proliferation and adhesion, indicating that MMA1 also plays a role in the malignant phenotype of melanoma cells. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. IgG Antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results demonstrated that FABP7 and MMA1 frequently expressed in melanoma may be potential targets for development of diagnostic and therapeutic methods.