Deubiquitinating enzyme OTUB2 increases the formation of neutrophil extracellular trapping nets and promotes the viability and invasion of colorectal cancer cells by inducing DDX54 activity
Objective The purpose of this study was to investigate the effects of OTU deubiquitinase,ubiquitin al-dehyde 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+DNase Ⅰ group(SW480 cells transfected with OTUB2 overexpression plasmid co-cultured with neutrophils treated with DNase Ⅰ),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 neutro-phils,respectively).ELISA was used to measure the relative expression of MPO-DNA complexes and the concen-tration of Cit-H3 in the supernatant of SW480 cells.MTT and Transwell assays were performed to evaluate cell via-bility 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 ex-periments.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 increased cell viability and invasion compared to the control group(P<0.05).In comparison to the vector group,the OTUB2 group showed increased relative ex-pression of MPO-DNA complexes and Cit-H3,as well as increased cell viability and invasion(P<0.05).Howev-er,when compared to the OTUB2 group,the OTUB2+DNase Ⅰ group exhibited decreased relative expression of MPO-DNA complexes and Cit-H3,as well as decreased cell viability and invasion(P<0.05).Co-IP,GST pull-down,and His-tag pull-down experiments demonstrated the interaction between OTUB2 and DDX54.In comparison to the OTUB2+si-NC group,the OTUB2+si-DDX54 group showed decreased relative expression of MPO-DNA complexes and Cit-H3,as well as decreased cell viability and invasion(P<0.05).Conclusion deubiquitinating enzyme OTUB2 can increase the formation of NETs and promote CRC cell viability and invasion by up-regulating DDX54.
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