The E3 Ubiquitin Ligase NEDD4 Promotes Lung Cancer Cell Proliferation And Migration Through The NF-κB Signaling

ObjectiveThis study is to explore the molecular mechanism of ubiquitin ligase NEDD4 in regulating lung cancer cell proliferation and migration through NF-κB signaling,thus further determine the role of NEDD4 in lung cancer development and progression,and provide new targeting pathways and molecules for targeted therapy of lung cancer.Methods1.The correlation between NEDD4 and multiple correlation factors of NF-κB signaling pathway in lung adenocarcinoma tissues was analyzed by UALCAN and GEPIA databases.2.NEDD4 knockdown in lung cancer A549 cell was constructed by sh RNA expression system with lentiviral vector,and NEDD4 knockdown effect was determined by Western Blot.3.The m RNA transcriptome of NEDD4 stable knockdown cell line(sh NEDD4)and control cell line(Vector)was analyzed by RNA-seq,compared differentially expressed genes between cell lines and identified a profile of NEDD4 knockdown-induced downregulated genes.4.NEDD4 knockdown cells and control cells were treated with IKK inhibitor BMS-345541,and the expression of NF-κB target genes CXCL1 and CXCL2 was detected by quantitative realtime PCR to determine whether NEDD4 regulates NF-κB signaling.5.The NF-κB transcriptional activity reporter plasmid p Green Fire1-NF-κB was expressed in NEDD4 knockdown cells and control cells,and the fluorescence intensity of green fluorescent protein(GFP)at basal and stimulated conditions was measured by flow cytometry to determine whether NEDD4 directly modulates NF-κB transcriptional activity.6.The level of the transcriptional repressor IκBα and transcriptional activator p65 in control and NEDD4 knockdown cells at basal level and under conditions of activating and inhibiting NF-κB signaling was detected by Western Blot,and treated with the proteasome inhibitor Velcade and the lysosome inhibitor chloroquine(CQ)to explore the molecular mechanism of NEDD4 in manipulating NF-κB signaling.7.The proliferation ability of NEDD4 knockdown cells and control cells was determined by cell counting assay,and the migration ability of NEDD4 knockdown cells and control cells was determined by scratch healing assay and Transwell assay,and combined with treatment of IKK inhibitor BMS-345541 to determine the effect of NEDD4 on regulating cell proliferation and migration through NF-κB signaling pathway.8.NEDD4 knockdown and control cells were treated with an inhibitor of CXC chemokine receptor 2(CXCR2)and an inhibitor of cyclooxygenase-2(COX-2),to determine whether NEDD4 promotes lung cancer cell proliferation and migration by regulating downstream target molecules of NF-κB,CXC chemokines and COX-2.Results1.Database analysis indicated that NEDD4 has high positive association with multiple positive correlation factors of NF-κB signaling pathway,suggesting that NEDD4 may be involved in modulating NF-κB signaling activation.2.Knockdown of NEDD4 in lung cancer A549 cell resulted in a significant downregulation of NF-κB downstream target molecules expression,especially the CXC chemokine family members(CXCL1,CXCL2,CXCL3,CXCL5,CXCL6,CXCL8)and cyclooxygenase-2(COX-2)were significantly decreased.3.With IKK inhibitor treatment,the gene expression of CXCL1/2 was markedly decreased in control cells,but that in NEDD4 knockdown cells was hardly changed,indicating that NEDD4 and IKK affected CXCL1/2 expression by mediating the same signaling pathway,or NEDD4 promoted CXCL1/2 expression by regulating NF-κB signaling.4.The average fluorescence intensity of GFP produced by the NF-κB transcriptional activity reporter plasmid in control cells was significantly higher than that in NEDD4 knockdown cells,suggesting that NEDD4 knockdown inhibited the transcriptional activity of NF-κB.5.There was no IκBα protein accumulation in control and NEDD4 knockdown cells upon the treatment of proteasome inhibitor.The IκBα protein in control cells was evidently increased after lysosome inhibitor treatment,but that in NEDD4 knockdown cells was hardly changed,suggesting that NEDD4 mediates lysosomal degradation of IκBα.6.NEDD4 knockdown significantly inhibited the proliferation and migration of lung cancer A549 cell.7.IKK inhibitor treatment dramatically inhibited the proliferation and migration of control cells,but had no significant inhibitory effect on NEDD4 knockdown cells,indicating that NEDD4 and IKK performed on the same signaling pathway to modulate lung cancer cell proliferation and migration.8.COX-2 inhibitor treatment significantly inhibited the proliferation and migration of control cells,but had no significant inhibitory effect on the migration of NEDD4 knockdown cells,these results suggested that COX-2,NF-κB downstream signaling molecule,partially regulates the promoting effect of NEDD4 on lung cancer cell proliferation and migration.Conclusions1.NEDD4 is a positive regulator of NF-κB signaling,which enhances NF-κB transcriptional activity by promoting lysosome-mediated degradation of IκBα,thereby promoting the expression of NF-κB downstream target genes.2.NEDD4 enhances the expression of NF-κB downstream target genes wihich are related with tumor proliferation and migration by elevating the transcriptional activity of NF-κB,then the products of these genes mediate lung cancer cell proliferation and migration,and among them,COX-2,a downstream target gene of NF-κB,is one of the downstream effectors.