AIM: To invesligale the role of HLA - F adjacent transcript 10 (FAT10) in tumorigenesis. METHODS; GAL4 yeast Lwo - hybrid assay was performed to screen the human Hep3B hepatoma cell cDNA library for obtaining the host cell prolein molecules which inleracl with FAT10. The immunofluoresence co - localization and co - im-munoprecipitation assay were applied to confirm the inleraclion of the idenlified proleins. FAT10 was knockdown by siRNA lo invesligale the changes of eukaryolic translation elongalion faclor 1 alpha 1 (eEFlAl) expression. RESULTS; The eEFlAl was selected from the hosl cells using yeasl lwo - hybrid assay. The results of co- localizalion and co - immuno-precipilalion assays further confirmed the inleraclion belween FAT10 and eEFlAl. Interestingly, when FAT10 was down -regulaled by siRNA in Hep3B cells, the prolein expression of endogenous eEFlAl was also found lo be significantly repressed. Furthermore, the expression of FAT10 was reduced by siRNA knockdown, thus resulting in the down - regulation of eEFlAl expression at both mRNA and prolein levels in MHCC97H cells. CONCLUSION: We propose a model in which eEFlAl serves as a substrale of FAT10 to accomplish, in part, its function in regulating the biological behaviors of tumor cells.%目的:利用酵母双杂交技术筛选类泛素蛋白FAT10(HLA-F adjacent transcript 10)在肝癌细胞Hep3B中相互作用的蛋白,为探讨FAT10蛋白在肝癌发生中的作用机制提供依据.方法:利用Clontech GAL4 酵母双杂交系统筛选人肝癌细胞系Hep3B的cDNA文库,以获得与FAT10相互作用的蛋白分子;通过回交实验、免疫共沉淀和激光共聚焦细胞内共定位实验验证两者之间的相互作用;通过RNA干扰降低FAT10表达观察真核翻译延伸因子1α1(eEF1A1)的表达情况.结果:酵母双杂交筛选得到相互作用蛋白eEF1A1,激光共聚焦细胞内共定位确定FAT10和eEF1A1均位于细胞质内,存在共定位;回交实验和免疫共沉淀实验再次确认eEF1A1能够与FAT10相互作用;FAT10表达降低伴随着eEF1A1的表达下调.结论:FAT10能够与eEF1A1相互作用;FAT10可能通过eEF1A1来实现其部分功能.
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