关键词: 痣和黑素瘤； 泛素缀合酶类； 肿瘤分期； 细胞系, 肿瘤； N-钙粘蛋白

Abstract: 【Abstract】 Objective To determine the expression of ubiquitin-conjugating enzyme E2S （UBE2S） in malignant melanoma （MM）, and to evaluate its effect on the biological behavior of melanoma cells. Methods Immunohistochemical study was performed to determine the UBE2S expression in 128 primary MM tissue chips, 64 metastatic MM tissue chips, 16 non-tumor tissue chips （8 paralesional normal skin tissues and 8 normal epidermal tissues）. Real-time quantitative RCR was conducted to determine the UBE2S mRNA expression in the melanoma cell lines A375, MUM-2B and MUM-2C. The melanoma cell lines A375 and MUM-2B were divided into 2 groups separately: interference group transfected with a lentiviral vector carrying UBE2S RNA interference sequence, and control group transfected with a lentiviral vector carrying control sequence. After 72 hours, real-time quantitative RCR was performed to determine the UBE2S mRNA expression in the melanoma cell lines A375 and MUM-2B. Caspase-3/7 activity in the groups was assessed by using kits, and cell apoptosis and cell cycle distribution were detected by flow cytometry. The effect of UBE2S knockdown on the migratory and invasive abilities of and N-cadherin expression in A375 cells were evaluated by Transwell assay and Western blot analysis respectively. Statistical analysis was carried out with SPSS 22.0 software by using independent sample t-test for the comparison of normally distributed data between two groups, chi-square test for enumeration data, Mann-Whitney U test for the comparison of non-normally distributed data, and Spearman′s coefficient for assessment of the correlation of UBE2S expression with T staging of melanoma. Results UBE2S was highly expressed in 98 （51.0%） MM tissues, but lowly expressed in 16 non-tumor tissues, and the UBE2S expression rate significantly differed between the above two kinds of tissues （χ2 = 11.905, P < 0.01）. UBE2S expression was negatively correlated with T staging of melanoma （ρ = -0.210, P = 0.043）. The relative mRNA expression of UBE2S significantly differed among the A375, MUM-2B, and MUM-2C cells （F = 817.228, P < 0.01）. After UBE2S knockdown, the caspase-3/7 activity was significantly up-regulated in the A375 interference group （t = 17.572, P < 0.01） and MUM-2B interference group （t = 24.552, P < 0.01） compared with the A375 and MUM-2B control groups respectively. Compared with the control group, the A375 interference group showed significantly increased proportion of A375 cells at G1 phase （t = 7.365, P < 0.01）, decreased proportion at S phase （t = -9.190, P < 0.01）, and no change in the proportion of A375 cells at G2/M phase （t = -0.227, P＞0.05）. The MUM-2B interference group showed significantly increased proportions of MUM-2B cells at G1 （t = 12.676, P < 0.01） and G2/M phases （t = 13.045, P < 0.01）, but significantly decreased proportion at S phase （t = -15.718, P < 0.01） compared with the control group. Transwell assay revealed decreased migratory and invasive abilities of A375 cells in the interference group compared with the control group （t = -35.727, -125.000, P < 0.05, < 0.01, respectively）. Western blot analysis showed down-regulated expression of N-cadherin protein in A375 cells in the interference group compared with the control group. Conclusions UBE2S is over-expressed in melanoma tissues, whose expression is associated with the T staging of melanoma. Knockdown of UBE2S affects the apoptosis, cell cycle, migration and invasion of melanoma cells, and may promote the metastasis of MM cells by regulating N-cadherin expression.

Key words: Nevi and melanomas, Ubiquitin?conjugating enzymes, Neoplasm staging, Cell line, tumor, N-cadherin