泛素连接酶Cullin3对三阴性乳腺癌细胞增殖、迁移和侵袭的影响及其机制探讨  

The effect of ubiquitin ligase Cullin3 on the proliferation,migration and invasion of triple-negative breast cancer cells and exploration of its mechanism

作　　者：何志贤[1] 李晶 王沁凡 程乾 倪苏婕 He Zhixian;Li Jing;Wang Qinfan;Cheng Qian;Ni Sujie(Department of Thyroid and Breast Surgery,Affiliated Hospital of Nantong University,Nantong 226001,China;Department of Electrocardiogram room,Affiliated Hospital of Nantong University,Nantong 226001,China;Department of Oncology,Affiliated Hospital of Nantong University,Nantong 226001,China)

机构地区：[1]南通大学附属医院甲乳外科,南通226001 [2]南通大学附属医院心电图室,南通226001 [3]南通大学附属医院肿瘤化疗科,南通226001

出　　处：《中华医学杂志》2024年第20期1868-1878,共11页National Medical Journal of China

基　　金：南通市卫生健康委科研课题(MS2023023);吴阶平医学基金会(320.6750.2023-18-24)

摘　　要：目的探讨泛素化连接酶E3蛋白(Cullin3,CUL3)对三阴性乳腺癌(TNBC)细胞增殖、迁移和侵袭能力的影响及其作用机制。方法基于生物信息学方法获取TNBC组织中CUL3基因和蛋白的表达数据,评估CUL3在TNBC患者肿瘤组织(n=160)中和正常乳腺组织(NC)(n=572)中的表达及其与临床预后的关系。通过CCK8细胞增殖实验、划痕实验、Transwell实验检测过表达CUL3对TNBC细胞体外增殖、迁移及侵袭能力的影响;通过免疫共沉淀(IP)联合质谱分析筛选出可能与CUL3相互作用的蛋白质,确定CUL3调控的底物蛋白为谷胱甘肽S-转移酶P1(GSTP1);通过划痕实验、Transwell实验检测过表达GSTP1对TNBC细胞迁移及侵袭能力的影响,探索过表达CUL3是否可以逆转GSTP1对细胞迁移及侵袭能力的影响;通过Western印迹和IP检测CUL3对GSTP1蛋白泛素化修饰的影响,验证CUL3调控GSTP1表达影响TNBC迁移和侵袭的分子机制。结果CUL3在TNBC中表达量显著增高(P<0.0001),CUL3高表达与TNBC患者预后不良相关,高表达CUL3的TNBC患者总生存期(OS)、无病生存期(RFS)均更短(OS,P=0.018;RFS,P=0.008);过表达CUL3可显著增加TNBC细胞增殖(231细胞组F=11.97,P=0.002;468细胞组F=51.92,P<0.001)、迁移[231细胞系和468细胞系过表达组穿膜细胞数分别为(74.7±4.0)、(128.0±6.1)个,而空白对照(NC)组分别为(21.0±2.7)、(70.0±6.6)个,231和468细胞组t分别为-19.24和-11.23,P均<0.001]和侵袭能力(231细胞系和468细胞系过表达组48 h细胞增殖率分别为56.6%±4.4%、51.6%±3.7%,而NC组分别为40.5%±2.9%、32.9%±4.8%,231细胞组t=-5.26,P=0.0063;468细胞组t=-5.38,P=0.0058);GSTP1在TNBC表达中降低,上调GSTP1可抑制TNBC细胞迁移[231细胞系和468细胞系过表达组穿膜细胞数分别为(16.3±6.5)、(33.0±6.2)个,而NC组分别为(34.3±2.5)、(77.3±5.0)个,231细胞组t=5.44,P=0.006;468细胞组t=7.20,P=0.002]和侵袭(231细胞系和468细胞系过表达组48 h细胞增殖率分别为49.6%±1.7%�Objective To investigate the effects of ubiquitin ligase Cullin3(CUL3)on the proliferation,migration and invasion ability of triple-negative breast cancer(TNBC)cells and its mechanism of action.Methods Bioinformatics-based methods were used to obtain CUL3 gene and protein expression data in TNBC tissues,and to assess the expression of CUL3 in tumour tissues of TNBC patients(n=160)and in normal breast tissues(n=572),and its relationship with clinical prognosis.The effects of overexpression of CUL3 on the proliferation,migration and invasion ability of TNBC cells in vitro were detected by CCK8 cell proliferation assay,scratch assay and transwell assay;proteins that might interact with CUL3 were screened by immunoprecipitation combined with mass spectrometry analysis,and the substrate protein regulated by CUL3 was identified as Glutathione S-Transferase Pi 1(GSTP1);the effects of overexpression of GSTP1 on the migration and invasion ability of TNBC cells were detected by scratch assay and Transwell assay,and it was explored whether overexpression of CUL3 could reverse the effects of GSTP1 on the migration and invasion ability of cells;and the effects of overexpression of GSTP1 on the migration and invasion ability of cells were detected by Western blot and IP(Immunoprecipitation)to detect the effect of CUL3 on the ubiquitination modification of GSTP1 protein,and to verify the molecular mechanism by which CUL3 regulates the expression of GSTP1 to affect TNBC migration and invasion.Results CUL3 expression was significantly higher in TNBC(P<0.0001),and high CUL3 expression was closely associated with poor prognosis of TNBC patients(OS,P=0.018;RFS,P=0.008);overexpression of CUL3 significantly increased the proliferation of TNBC cells(F=11.97,P=0.002 for the 231-cell group,F=51.92,P<0.001 for the 468-cell group),migration[74.7±4.0 and 128.0±6.1 perforating cells in the overexpression groups of 231 and 468 cell lines,compared with 21.0±2.7 and 70.0±6.6 in the blank control(NC)group,and the t-values of 231 and 468 cell

关 键 词：乳腺肿瘤 Cullin蛋白质类 谷胱甘肽S-转移酶P1 泛素化 

分 类 号：R737.9[医药卫生—肿瘤]