| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 104 === Nucleotide diphosphate kinase A (NDPK-A) is encoded by the nm23-H1/nme1 gene, and acts as a metastasis promoter in neuroblastoma. Overexpression or S120G mutation of NDPK-A found in advanced neuroblastoma increases both invasive and metastatic potential of neuroblastoma cells. However, the molecular mechanism by which NDPK-A promotes neuroblastoma metastasis remains poorly understood. Our lab has found a novel protein, termed Protein Y, that interacted with NDPK-A, but not NDPK-AS120G. In this study, non-sense mutations of the Protein Y mRNA were found in human neuroblastoma NB69, IMR32 and SH-SY5Y cell lines. In stable transducents established from NB69 cells, ectopic expression of NDPK-A or NDPK-AS120G increased cell migration by more than two folds, whereas ectopic expression of Protein Y decreased cell migration by 47% in the trans-well assay. Co-expression of Protein Y completely abolished the migration-enhancing ability of NDPK-A. In contrast, Protein Y displayed little effect on the migration-enhancing ability of NDPK-AS120G. In the clonogenic assay, Protein Y reduced the colony number by 17%, whereas NDPK-A or NDPK-AS120G increased the colony number by 20-25%. Co-expressed Protein Y almost completely abolished the colony-enhancing ability of NDPK-A, but not NDPK-AS120G. While knocking down Protein Y expression partially restored the invasiveness-enhancing ability of NDPK-A, deletion of the N-terminus abolished the ability of Protein Y to counteract the invasiveness-enhancing ability of NDPK-A and, to a lesser degree, NDPK-AS120G. In a zebrafish transplantation model, Protein Y did not seem to affect the extravasation ability of NB69 cells, nor suppress the extravasation-enhancing ability of NDPK-AS120G. By whole mount in-situ hybridization, zebrafish NDPK-A and Protein Y orthologs, namely nme2b.2 and Protein Y, did not appear to participate in the differentiation of neural crest into sympathetic neurons, relevant to neuroblastoma. In conclusion, Protein Y counteracted the ability of NDPK-A and, to a lesser degree, NDPK-AS120G in promoting the invasiveness of NB69 cells. However, such a counteracting ability of Protein Y is likely abrogated by mutation-caused truncations, as detected in its mRNA in human neuroblastoma cell lines examined.
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