泛素结合酶2S在恶性黑素瘤中的表达及其对黑素瘤细胞生物学行为的影响  

Expression of ubiquitin-conjugating enzyme E2S in malignant melanoma and its effect on the biological behavior of melanoma cells

作　　者：马阳阳 王平[2] 胡文婷 徐永平 张小燕[2] 黎钊[2] 虞闻仲 朱蒙燕 许爱娥[2] Ma Yangyang;Wang Ping;Hu Wenting;Xu Yongping;Zhang Xiaoyan;Li Zhao;Yu Wenzhong;Zhu Mengyan;Xu Ai′e(Zhejiang Chinese Medical University,Hangzhou 310009,China;Department of Dermatology,Hangzhou Third Hospital,Hangzhou 310009,China;Department of Dermatology,Yiwu Dermatology Hospital,Yiwu 322000,Zhejiang,China)

机构地区：[1]浙江中医药大学,杭州310009 [2]杭州市第三人民医院皮肤科,310009 [3]浙江省义乌市皮肤病医院皮肤科,322000

出　　处：《中华皮肤科杂志》2019年第8期542-547,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81472887);浙江省省级公益技术应用研究计划项目(2016C33206);浙江省医药卫生科技计划项目(2014KYB200);义乌市一般科研计划项目(17-1-29).

摘　　要：目的探讨恶性黑素瘤(MM)组织中泛素结合酶2S(UBE2S)的表达及其对黑素瘤细胞生物学行为的影响。方法应用免疫组化检测UBE2S在128例原发性MM、64例转移性MM、16份非瘤组织(8份瘤旁组织、8份正常表皮组织)芯片中的表达。实时定量PCR检测A375、MUM-2B及MUM-2C黑素瘤细胞株中UBE2S mRNA表达水平。将黑素瘤A375及MUM-2B细胞株分别分为两组,干扰组使用含有UBE2S RNA干扰序列的慢病毒转染,对照组使用含有对照序列的慢病毒转染,72 h后实时定量PCR检测A375和MUM-2B黑素瘤细胞株中UBE2S mRNA表达水平。用试剂盒检测各组细胞caspase3/7活性,流式细胞仪检测各组细胞凋亡率及周期分布;应用Transwell法及Western印迹分别检测敲减UBE2S对A375细胞迁移侵袭能力及N-钙黏蛋白表达的影响。使用SPSS 22.0软件进行统计学分析,正态分布数据组间比较采用独立样本t检验,计数资料采用卡方检验,非正态分布数据比较采用Mann-Whitney U检验,通过Spearman系数评估UBE2S表达水平与T分期的相关性。结果UBE2S在98例(51.0%)MM组织中高表达,16例非瘤组织中均低表达,两组间高表达率差异有统计学意义(χ^2 = 11.905,P<0.01);UBE2S表达水平与肿瘤T分期呈负相关(ρ=-0.210,P = 0.043)。A375、MUM-2B、MUM-2C细胞间UBE2S mRNA相对表达量差异有统计学意义(F = 817.228,P<0.01)。Caspase3/7活性在干扰组A375细胞(t = 17.572,P<0.01)与干扰组MUM-2B细胞(t = 24.552,P<0.01)分别较相应对照组明显增加;干扰组A375细胞较对照组G1期细胞比例明显升高(t = 7.365,P<0.01),而S期比例明显降低(t =-9.190,P<0.01),G2/M期无明显变化(t =-0.227,P>0.05);干扰组MUM-2B细胞G1期(t = 12.676,P<0.01)、G2/M期(t = 13.045,P<0.01)细胞比例高于对照组,但S期比例低于对照组(t =-15.718,P<0.01)。Transwell实验显示,干扰组较对照组A375细胞迁移(t =-35.727,P<0.05)及侵袭(t =-125.000,P<0.01)能力降低。Western印迹检测显示,干扰组较对照组A3Objective 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 (χ^2 = 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 differe

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 

分 类 号：R739.5[医药卫生—肿瘤]