Fig. 4

NETs triggered an enhanced metastasis potential by raising an inflammatory response headed by COX2 elevation. a Venn diagram of the common up/downregulated genes detected by RNA-seq in HepG2/MHCC97H cells treated with NETs. b Heatmap of altered genes and the GO biological process analysis. COX2 and other pro-inflammatory genes were robustly upregulated in response to NETs. c MeV software of protein network predicting COX2 at the central of NET-induced inflammatory response (http://string.embl.de/). d RT-PCR verification of upregulation of COX2 expression in HepG2 and MHCC97H in response to NETs, but not intact neutrophils or NETs with DNase 1 and boil abrogation. e Immunofluorescence assay verifying the upregulation of COX2 expression in HepG2 and MHCC97H cells. f Western blot showing upregulation of COX2 correlated with NET marker H3cit in metastasis liver lesions from LPS-induced NETs mice. g Representative images of higher COX2 expression in NET-high HCC tissues (identified by median of H3cit staining in Fig. 1h, n = 104) and statistic graph. Scare bar: 50 μm. h Pearson correlation analysis of NETs and COX2 expression in HCC samples (n = 104). i Follow-up data of the 88 non-metastasis/recurrence HCC patients with different NET and COX2 expression for metastasis/recurrence cumulative incidence analysis. Log-rank test was used. j An extruded cytotoxicity of NETs on cell death rate of HepG2/MHCC97H cells pretreated with COX2 inhibitor Celecoxib in TUNEL assay. k An inhibitory effect of Celecoxib on invasion capacity of HepG2/MHCC97H cells treated by NETs. l No change in early adhesion number of Celecoxib-treated hepa1-6 cells in LPS-induced NET mice. Dil-labeled Hepa1-6 cells were pretreated with Celecoxib followed by intraspleen injection, and liver frozen sections 2 h post-injection were observed under fluorescence microscope for early adhesion quantification (n = 5 each). m Less metastasis lesions formed by Celecoxib-treated hepa1-6 cells in LPS-induced NET mice model (n = 5 each). Saline served as control. *P < 0.05; **P < 0.01; ***P < 0.001. Data were presented as means ± SEM