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是重要的。通过使用GeneChip比较黑素瘤细胞系与各种正常组织的表达谱，并通过RT-PCR确认所选基因的实际表达，我们鉴定了FABP 7、MMA 1和KU-MEL-3。通过RNA干扰和重组转导分析这些基因的功能。通过用FABP 7特异性siRNA下调FABP 7表达，在黑色素瘤细胞系中抑制体外细胞增殖和Matrigel侵袭。通过用FABP 7 cDNA转染在FABP 7阴性的胚肾细胞系293 T中过表达FABP 7，导致增强的细胞增殖和Matrigel侵袭，表明FABP 7在一些黑素瘤细胞系的恶性表型中起作用。MMA 1特异性siRNA下调MMA 1可抑制黑色素瘤细胞系的体外细胞增殖和细胞粘附。通过用MMA 1重组体转染，在具有低MMA 1表达的黑素瘤细胞系中MMA 1的过表达导致增强的细胞增殖和粘附，表明MMA 1在黑素瘤细胞的恶性表型中也起作用。免疫组化结果显示，FABP 7在15例黑色素瘤组织中有11例表达。分别在来自黑素瘤患者的25份血清中的14份（56%）或31份血清中的8份（26%）中检测到对噬菌体或细菌重组FABP 7蛋白特异性的IgG抗体，但在来自健康个体的血清中未检测到，表明FABP 7是黑素瘤患者中的免疫原性抗原。这些结果表明，FABP 7和MMA 1在黑色素瘤中频繁表达，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.