TRIM59通过结合BCLAF1调控人皮肤黑色素瘤SK-MEL-2细胞的恶性生物学行为  

TRIM59 regulates malignant biological behaviors of skin cutaneous melanoma cells SK-MEL-2 through combination with BCLAF1

作　　者：刘建敏[1] 周亚净[1] 何润之[1] 段春胜[1] LIU Jianmin;ZHOU Yajing;HE Runzhi;DUAN Chunsheng(Department of Hand and Foot Surgery,Xingtai People's Hospital,Xingtai 054001,Hebei,China)

机构地区：[1]邢台市人民医院手足外科,河北邢台054001

出　　处：《中国肿瘤生物治疗杂志》2022年第8期724-731,共8页Chinese Journal of Cancer Biotherapy

基　　金：邢台市重点研发计划资助项目(No.2020ZC233)。

摘　　要：目的:探究三结构域蛋白59(TRIM59)调控人皮肤黑色素瘤细胞SK-MEL-2增殖、细胞周期、凋亡及迁移侵袭的作用机制,及其与Bcl2相关转录因子1(BCLAF1)之间的关系。方法:qPCR和WB法检测人表皮黑色素细胞HEMn-LP、人皮肤黑色素瘤细胞SK-MEL-2、UACC903、A375及36例邢台市人民医院2019年2月至2021年7月收集的皮肤黑色素瘤组织中TRIM59的mRNA和蛋白表达,使用脂质体将si-con、si-TRIM59转染至SK-MEL-2细胞中,WB法检测干扰TRIM59表达对细胞中周期蛋白D1(CCND1)、细胞周期素依赖性激酶2(CDK2)、肿瘤抑制蛋白基因(TP53)和BCLAF1蛋白表达的影响,CCK-8法、流式细胞术、划痕愈合实验、Transwell实验检测对细胞的活性、凋亡、迁移和侵袭的影响,免疫共沉淀(Co-IP)实验检测对细胞中TRIM59蛋白与BCLAF1结合能力的影响。结果:与HEMn-LP细胞相比,SK-MEL-2、UACC903、A375细胞中TRIM59 mRNA和TRIM59、BCLAF1蛋白均呈高表达(均P<0.05),SK-MEL-2细胞中TRIM59表达水平最高。相较于si-con组和Normal组,沉默TRIM59后,SK-MEL-2细胞的活性显著降低,细胞周期阻滞于G2期,CCND1、CDK2的蛋白表达显著降低,TP53蛋白和细胞凋亡率均显著升高,划痕抑制率明显升高,迁移侵袭细胞数明显降低(均P<0.05)。免疫共沉淀实验结果显示,TRIM59与BCLAF1之间存在蛋白结合关系。TRIM59与BCLAF1在肿瘤组织中的表达呈显著的正相关(r=0.878,P<0.001)。结论:干扰TRIM59表达能够抑制人皮肤黑色素瘤SK-MEL-2细胞的增殖、迁移和侵袭而促进凋亡,抑制SK-MEL-2细胞的恶性生物学行为,其机制可能与TRIM59结合BCLAF1有关。Objective:To explore the mechanism of tripartite motif-containing 59(TRIM59)regulating the proliferation,cell cycle,apoptosis,migration and invasion of human skin melanoma cells SK-MEL-2,and its relationship with Bcl2-associated transcription factor(BCLAF1).Methods:qPCR and WB assay were used to measure the mRNA and protein expression of TRIM59 in human epidermal melanocytes HEMN-LP,human skin melanoma cells SK-MEL-2,UACC903,A375,and 36 cases of human skin melanoma tissues collected from February 2019 to July 2021 in Xingtai people’s Hospital.Si-con and si-TRIM59 were transfected into SK-MEL-2cells using liposomes.WB assay was used to detect the effects of interference with the expression of TRIM59 on cyclin D1(CCND1),cyclin-dependent kinase 2(CDK2),tumor suppressor protein gene(TP53)and BCLAF1 protein expression.CCK-8 assay,flow cytometry,scratch test and Transwell test were used to detect cell activity,apoptosis,migration and invasion.The binding ability of TRIM59 protein and BCLAF1 was detected by Co-IP assay.Results:Compared with the HEMN-LP group,the mRNA and protein expression of TRIM59 BCLAF1 protein in SK-MEL-2,UACC903and A375 cells were significantly increased(P<0.05).The expression level of TRIM59 in SK-MEL-2 cells were the highest.Compared with the si-con group and the Normal group,after silencing TRIM59,the activity of SK-MEL-2 cells was significantly decreased;the G2 phase of the cell cycle was blocked;the protein expressions of CCND1 and CDK2 were decreased significantly;the TP53 protein and apoptosis rate were significantly increased;the scratch inhibition rate was significantly increased,and the numbers of migration and invasion cells were significantly decreased.(all P<0.05).The results of co-immunoprecipitation experiments showed that there was a protein-binding relationship between TRIM59 and BCLAF1.There was a significant positive correlation between TRIM59 and BCLAF1 expression in tumor tissues(r=0.878,P<0.001).Conclusion:Silencing TRIM59 expression could inhibit the proliferation,migration

关 键 词：皮肤黑色素瘤 SK-MEL-2细胞 TRIM59 BCLAF1 蛋白结合 恶性生物学行为 

分 类 号：R739.5[医药卫生—肿瘤] R730.2[医药卫生—临床医学]