肿瘤坏死因子受体相关因子6第331位泛素化位点突变对结直肠癌细胞生物学特征的影响及其机制  

Construction of TRAF6 ubiquitin site 331 mutant colorectal cancer cell stable line and its effect on biological behavior of colorectal cancer cells

作　　者：何若凡 王琴 林春霖[1] 林鹏航 陈辉 黄永建[1] 杨树钢[1] 叶建新[1] 朱广伟[1] He Ruofan;Wang Qin;Lin Chunlin;Lin Penghang;Chen Hui;Huang Yongjian;Yang Shugang;Ye Jianxin;Zhu Guangwei(Department of Gastrointestinal Surgery 2 Section,the First Hospital Affiliated to Fujian Medical University,Institute of Abdominal Surgery,Key Laboratory of Accurate Diagnosis and Treatment of Cancer,Fuzhou 350005,China;Key Laboratory of Ministry of Education for Gastrointestinal Cancer,Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院胃肠外科二区,腹部外科研究所,肿瘤精准诊疗重点实验室,福州350005 [2]福建医科大学消化道恶性肿瘤教育部重点实验室,福州350005

出　　处：《中华肿瘤杂志》2023年第2期129-137,共9页Chinese Journal of Oncology

基　　金：国家自然科学基金(81702424、81872364);福建省卫健委创新课题(2020CXA032)。

摘　　要：目的探讨肿瘤坏死因子受体相关因子6(TRAF6)第331位泛素化位点突变对结直肠癌细胞生物学特征的影响及其相关机制。方法将带有绿色荧光蛋白标签的TRAF6基因表达质粒的慢病毒(pCDH-3×FLAG-TRAF6)和突变质粒慢病毒(pCDH-3×FLAG-TRAF6-331mut)分别感染结直肠癌细胞SW480和HCT116,荧光显微镜观察细胞感染情况,Western blot检测TRAF6和TRAF6-331mut在细胞中的表达。采用细胞计数试剂盒8法和平板克隆实验检测TRAF6组和TRAF6-331mut组结直肠癌细胞的增殖能力,细胞划痕实验检测细胞的迁移能力,Transwell小室实验检测细胞的迁移和侵袭能力,免疫共沉淀方法检测TRAF6和TRAF6-331mut与泛素的赖氨酸结合位点K48和K63的泛素化作用,Western blot检测过表达TRAF6和TRAF6-331mut对核因子κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)/激活蛋白1(AP-1)信号通路的影响。结果荧光显微镜下观察到病毒成功感染结直肠癌细胞。Western blot检测显示,TRAF6和TRAF6-331mut在结直肠癌细胞中成功表达。CCK-8法检测结果显示,到第4天,TRAF6-331mut组HCT116和SW480细胞的A值分别为1.89±0.39和1.88±0.24,均低于TRAF6组(分别为2.09±0.12和2.17±0.45,P值分别为0.036和0.011)。平板克隆形成实验结果显示,TRAF6-331mut组HCT116和SW480细胞的克隆数分别为(120±14)个和(85±14)个,均低于TRAF6组[分别为(190±21)个和(125±13)个,P值分别为0.001和0.002]。细胞划痕实验结果显示,48 h后,TRAF6-331mut组HCT116和SW480细胞的伤口愈合距离百分比分别为(31±12)%和(33±14)%,均低于TRAF6组[分别为(43±13)%和(43±7)%,P值分别为0.005和0.009]。Transwell迁移实验结果显示,TRAF6-331mut组HCT116和SW480细胞的迁移数分别为(104±13)个和(107±12)个,均低于TRAF6组[分别为(172±19)个和(138±16)个,P值分别为<0.001和0.002]。Transwell侵袭实验结果显示,TRAF6-331mut组HCT116和SW480细胞的穿膜数分别为(103±17)个和(92±13)个,均低于TRAF6组[分别为(177�Objective To investigate the effect of ubiquitin mutation at position 331 of tumor necrosis factor receptor related factor 6(TRAF6)on the biological characteristics of colorectal cancer cells and its mechanism.Methods lentivirus wild type(pCDH-3×FLAG-TRAF6)and mutation(pCDH-3×FLAG-TRAF6-331mut)of TRAF6 gene expression plasmid with green fluorescent protein tag were used to infect colorectal cancer cells SW480 and HCT116,respectively.The infection was observed by fluorescence microscope,and the expressions of TRAF6 and TRAF6-331mut in cells was detected by western blot.Cell counting kit-8(CCK-8)and plate cloning test were used to detect the proliferation ability of colorectal cancer cells in TRAF6 group and TRAF6-331mut group,cell scratch test to detect cell migration,Transwell chamber test to detect cell migration and invasion,immunoprecipitation to detect the ubiquitination of TRAF6 and TRAF6-331mut with ubiquitinof lysine binding sites K48 and K63.Western blot was used to detect the effects of TRAF6 and TRAF6-331mut over expression on the nuclear factor kappa-B(NF-κB)and mitogen activated protein kinase mitogen-activated protein kinase(MAPK)/activating protein-1(AP-1)signal pathway.Results The successful infection of colorectal cancer cells was observed under fluorescence microscope.Western blot detection showed that TRAF6 and TRAF6-331mut were successfully expressed in colorectal cancer cells.The results of CCK-8 assay showed that on the fourth day,the absorbance values of HCT116 and SW480 cells in TRAF6-331mut group were 1.89±0.39 and 1.88±0.24 respectively,which were lower than those in TRAF6 group(2.09±0.12 and 2.17±0.45,P=0.036 and P=0.011,respectively).The results of plate colony formation assay showed that the number of clones of HCT116 and SW480 cells in TRAF6-331mut group was 120±14 and 85±14 respectively,which was lower than those in TRAF6 group(190±21 and 125±13,P=0.001 and P=0.002,respectively).The results of cell scratch test showed that after 48 hours,the percentage of wound healing dis

关 键 词：结直肠肿瘤 肿瘤坏死因子受体相关因子6 泛素化 突变 核因子ΚB 激活蛋白1 

分 类 号：R735.34[医药卫生—肿瘤]