UBA3在黑素瘤细胞的表达及其影响黑素瘤细胞生物学行为的研究

Expression of ubiquitin-like modifier activating enzyme 3 in melanoma cells and its effect on the biological behavior of melanoma cells

目的 探讨UBA3在黑素瘤细胞中的表达，初步观察干扰UBA3对黑素瘤细胞生物学行为的影响。 方法 用RT-PCR技术观察3株黑素瘤细胞中UBA3的表达，并利用siRNA技术对M14细胞中UBA3的表达进行干扰，检测转染后细胞内UBA3、连接态NEDD8以及p53的表达，并观察转染前后细胞的凋亡及迁移能力的改变。 结果 在A375、M14及MV3中UBA3的表达均较正常黑素细胞上调，其中M14细胞上调最明显，成功干扰M14中UBA3的表达后，细胞内连接态NEDD8的表达下降，p53的表达明显增加，同时M14细胞凋亡增加、迁移能力下降。 结论 构建的质粒可干扰UBA3的表达，影响细胞的Nedd化进程，从而影响细胞的生物学行为。

Objective To detect the expression of ubiquitin-like modifier activating enzyme 3 （UBA3） in melanoma cells, and to estimate the effect of RNA interference in UBA3 expression on the biological behavior of melanoma cells. Methods Reverse transcription-polymerase chain reaction （RT-PCR） was performed to detect the expression of UBA3 in three melanoma cell lines （A375, M14 and MV3） and normal melanocytes from healthy human foreskin. Three short hairpin RNAs （shRNAs） targeting the UBA3 gene were designed and synthesized. To select the most efficient shRNA, some M14 cells were transfected with constructed plasmids containing the three shRNAs separately and an empty plasmid respectively followed by the determination of UBA3 and NEDD8 （neural precursor cell expressed, developmentally down-regulated 8） expression by Western blot. Then, some M14 cells were divided into two groups to be transfected with the selected shRNA-containing plasmid （shRNA-UBA3） and an empty plasmid respectively, with untransfected cells serving as the control. Subsequently, the expression of p53 was measured by Western blot, cell apoptosis detected by Hoechest 33258 nuclear staining, and migration activity evaluated by Transwell assay. Results The expression of UBA3 was significantly up-regulated in all the three melanoma cell lines, especially in M14 cells, compared with the normal melanocytes. In comparison with the M14 cells remaining untransfected and transfected with the empty plasmid, those transfected with shRNA-UBA3 showed an obvious decrease in the expression of NEDD8 ligated to proteins and migratory activity （all P 〈 0.05）, but an increase in p53 expression and cell apoptosis. Conclusions The shRNA-UBA3 eukaryotic expression plasmid constructed in this study could efficiently interfere in the expression of UBA3, affect the neddylation process, and influence the biological behavior of M14 cells.