摘要
目的探讨含OTU结构域的泛素醛结合蛋白2(OTUB2)影响RNA解螺旋酶54(DDX54)的活性及其对中性粒细胞胞外诱捕网(NETs)的形成和结直肠癌(CRC)细胞活力、侵袭的影响。方法分离CRC患者和健康对照组的外周血中性粒细胞,并使用佛波酯(PMA)或脱氧核糖核酸酶Ⅰ(DNaseⅠ)刺激中性粒细胞4 h;Western blot检测NETs标记物髓过氧化物酶(MPO)和瓜氨酸组蛋白H3(Cit-H3)的表达;干预SW480细胞中OTUB2或DDX54的表达并将其和中性粒细胞共培养,细胞分为如下组:对照组(不做任何处理),NETs组、vector组、OTUB2组、NETs+si-OTUB2组(SW480细胞、转染了vector、OTUB2过表达质粒和si-OTUB2的SW480细胞分别与PMA处理的中性粒细胞共培养),OTUB2+DNaseⅠ组(转染了OTUB2过表达质粒的SW480细胞与DNaseⅠ处理的中性粒细胞共培养),si-OTUB2组、OTUB2+si-NC组和OTUB2+si-DDX54组(转染了si-OTUB2、OTUB2过表达质粒和si-NC、OTUB2过表达质粒和si-DDX54的SW480细胞分别与中性粒细胞共培养)。ELISA检测SW480细胞上清液中MPO-DNA复合物相对表达量和Cit-H3的浓度;MTT和Transwell检测SW480细胞活力和侵袭能力;RNA测序筛选OTUB2调控的下游基因并使用实时荧光定量聚合酶链反应(qRT-PCR)、免疫共沉淀(co-IP)和谷胱甘肽S-转移酶(GST)亲和纯化(GST-pull-down)、His-tag pull-down实验验证DDX54和OTUB2的相互作用。结果与健康对照相比,CRC患者外周血中NETs的形成增加。与对照组相比,NETs组CRC细胞活力和侵袭增加(P<0.05)。与vector组比较,OTUB2组MPO-DNA复合物相对表达量和Cit-H3的浓度增加,细胞活力和侵袭增加(P<0.05);而与OTUB2组比较,OTUB2+DNaseⅠ组MPO-DNA复合物相对表达量和Cit-H3的浓度减少,细胞活力和侵袭减少(P<0.05)。Co-IP实验、GST pull down实验和His-tag pull down实验表明OTUB2和DDX54之间存在相互作用。与OTUB2+si-NC组比较,OTUB2+si-DDX54组MPO-DNA复合物相对表达量和Cit-H3的浓度减少,细胞活力和侵袭减少(P<0.
Objective The purpose of this study was to investigate the effects of OTU deubiquitinase,ubiquitin aldehyde binding 2(OTUB2)on the activity of DEAD-box helicase 54(DDX54)and its influence on the formation of neutrophil extracellular traps(NETs)and the vitality and invasion of colorectal cancer(CRC)cells.Methods Peripheral blood neutrophils were isolated from CRC patients and healthy controls,then stimulated with phorbol-12-myristate-13-acetate(PMA)or DNaseⅠfor 4 hours.Western blot analysis was performed to detect the expression of NETs markers,myeloperoxidase(MPO),and citrullinated histone H3(Cit-H3).The expression of OTUB2 or DDX54 was manipulated in SW480 cells,and they were co-cultured with neutrophils.The cells were divided into the following groups:Control group(without any treatment),NETs group,vector group,OTUB2 group,NETs+si-OTUB2 group(SW480 cells,SW480 cells transfected with vector,OTUB2 overexpression plasmid and si-OTUB2 were co-cultured with PMA-treated neutrophils,respectively),OTUB2+DNaseⅠgroup(SW480 cells transfected with OTUB2 overexpression plasmid co-cultured with neutrophils treated with DNaseⅠ),si-OTUB2group,OTUB2+si-NCgroup,OTUB2+si-DDX54group(SW480 cells transfected with si-OTUB2,OTUB2 overexpressing plasmid and si-NC,OTUB2 overexpressing plasmid and si-DDX54 were co-cultured with neutrophils,respectively).ELISA was used to measure the relative expression of MPO-DNA complexes and the concentration of Cit-H3 in the supernatant of SW480 cells.MTT and Transwell assays were performed to evaluate cell viability and invasive ability.RNA sequencing was conducted to screen downstream genes regulated by OTUB2.The interaction between DDX54 and OTUB2 was validated using quantitative real-time polymerase chain reaction(qRT-PCR),co-immunoprecipitation(Co-IP),Glutathione S-Transferase(GST)pull-down,and His-tag pull-down experiments.Results Compared to healthy individuals,there was an increased formation of NETs in the peripheral blood of CRC patients.The CRC cells in the NETs group exhibited increa
作者
蒋良君
李卫
Jiang Liangjun;Li Wei(Gastroenterology,The Affiliated Nanhua Hospital,Hengyang Medical School,University of South China,Hengyang 421002;Hepatobiliary surgery,The Affiliated Nanhua Hospital,Hengyang Medical School,University of South China,Hengyang 421002)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第3期463-472,共10页
Acta Universitatis Medicinalis Anhui
基金
湖南省卫生健康委卫生科研项目(编号:D202303038378)。
