Abstract: Cheng Fang*, He Runzhi, Zhang Lei, Zhang Wei, Gao Wei, Lyu Jing, Sun Jianfang. *Xingtai People′s Hospital, Xingtai 054001, Hebei, China Corresponding author: Sun Jianfang, Email: fangmin5758@aliyun.com 【Abstract】 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.

Key words: Melanoma, Ubiquitin, Ubiquitin-like modifier activating enzyme 3, Apoptosis, Cell movement