泛素连接酶E4B促进结直肠癌耐药机制的研究  

The study of ubiquitin ligase E4B in promoting drug resistance mechanisms in colorectal cancer

作　　者：朱秀清 王永强 周春丽 Zhu Xiuqing

机构地区：[1]浙江新安国际医院,314000 [2]浙江省嘉兴市第二医院,314000

出　　处：《浙江临床医学》2025年第2期200-202,共3页Zhejiang Clinical Medical Journal

基　　金：嘉兴市秀洲区科技计划项目(2022C001);嘉兴市科技计划项目(2021AD30099)。

摘　　要：目的探讨泛素连接酶E4B(UBE4B)对结直肠癌细胞多药耐药的影响。方法建立人结直肠癌耐药细胞株Caco-2/MDR,体外培养,并设置对照组、空载质粒组、UBE4B过表达组、PTCH1过表达组和联合过表达组。培养48 h后,加入奥沙利铂与5-Fu,7 d后进行检测。采用RT-PCR测定UBE4B和PTCH1 mRNA表达水平;利用蛋白免疫印迹实验测定hedgehog信号通路成员、下游细胞周期以及凋亡相关蛋白的表达水平;使用细胞增殖和CCK-8测定Caco-2/MDR细胞的增殖水平;采用annexin V与PI染色测定细胞凋亡率;Transwell细胞侵袭实验、细胞划痕实验与平板克隆实验测定细胞的侵袭、迁移与克隆形成能力;利用免疫共沉淀和泛素化实验测定UBE4B和PTCH1的相互作用。采用双因素方差分析比较药物干预前后各指标的差异。结果联合过表达组的PTCH1表达水平显著低于PTCH1过表达组,UBE4B表达水平显著高于PTCH1过表达组。联合过表达组的细胞增殖活性、迁移、侵袭与克隆形成能力均显著高于PTCH1过表达组,低于UBE4B组。而联合过表达组的细胞凋亡率则呈现出相反的趋势。药物干预后,UBE4B组的细胞增殖活性、迁移、侵袭与克隆形成能力均显著高于对照组,联合表达组的上述指标与对照组相比差异无统计学意义。UBE4B与PTCH1存在相互作用,二者的表达水平呈负相关。UBE4B可介导PTCH1的泛素化。结论UBE4B能够引发结直肠癌细胞的多药耐药,其机制可能与UBE4B介导的PTCH1泛素化对Hedgehog信号通路的调控有关。Objective To investigate the impact of ubiquitin factor E4B(UBE4B)on the multidrug resistance of colorectal cancer cells.Methods A human colorectal cancer drug-resistant cell line,Caco-2/MDR,was established and cultured in vitro,with the setting of the control group,the empty vector group,the UBE4B overexpression group,the PTCH1 overexpression group,and the combined overexpression group.After 48 hours of culture,oxaliplatin and 5-Fu were added,and the cells were tested after 7 days.Quantitative PCR(RT-PCR)was used to determine the mRNA expression levels of UBE4B and PTCH1.Protein immunoblotting was utilized to measure the expression levels of hedgehog signaling pathway members,downstream cell cycle,and apoptosis-related proteins.A cell proliferation and cytotoxicity detection kit(CCK-8)was employed to assess the proliferation levels of Caco-2/MDR cells.Annexin V and propidium iodide(PI)staining was applied to determine the rate of cell apoptosis.Transwell cell invasion assay,cell scratch assay,and clonogenic assay were conducted to evaluate cell invasion,migration,and clonogenic abilities.Immunoprecipitation and ubiquitination assays were used to determine the interaction between UBE4B and PTCH1.Two-way ANOVA was applied to compare the differences in various indicators among the groups before and after drug intervention.Results The PTCH1 expression level in the combined overexpression group was significantly lower than in the PTCH1 overexpression group,while the UBE4B expression level was significantly higher than in the PTCH1 overexpression group.The cell proliferation activity,migration,invasion,and clonogenic abilities in the combined overexpression group were significantly higher than in the PTCH1 overexpression group but lower than in the UBE4B group.Conversely,the apoptosis rate in the combined overexpression group showed an opposite trend.After drug intervention,the cell proliferation activity,migration,invasion,and clonogenic abilities in the UBE4B group were significantly higher than in the control group

关 键 词：E3泛素连接酶 晚期结直肠癌 多药耐药 泛素化 HEDGEHOG信号通路 

分 类 号：R73[医药卫生—肿瘤]