Neutrophil Extracellular Traps Induced By Interleukin 8 Via CXC Chemokine Receptor 1/2 Promote The Progression Of Gastric Carcinoma Through Transcription Factor ⅡB-related Factor 1

Background Gastric cancer（GC）is one of the most common cancers in China,and research on the mechanism of its occurrence and progression and improvement of poor prognosis,has never stopped.Neutrophil Extracellular traps（NETs）was proved to not only sequester pathogens in inflammatory conditions but also neoplastic cells in the context of cancer,playing a role in tumor growth and progression,angiogenesis,metastasis and tumor-associated thrombosis.Our preliminary study had shown that neutrophils and NETs were infiltrated in the tissue microenvironment and peripheral blood of GC patients.NETs had novel diagnostic,therapeutic predictive,and prognostic value in GC patients.IL-8 mediated NETosis by targeting CXC chemokine receptor 1/2（CXCR1/2）in other tumors,but there is no study on the upstream regulation mechanism of NETs in GC.In addition,our preliminary study had also shown that GC tissues with high expression of transcription factor IIB-related factor 1（BRF1）and MPO-positive neutrophils infiltration were related to poor prognosis of GC patients.Whether NETs in the microenvironment is related to the expression of BRF1 in GC cells,thus promoting tumorigenesis,deserves further exploration.Methods Human neutrophils were isolated using density gradient centrifugation.Interleukin 8（IL-8）,Phorbol-12-myristate-13-acetate（PMA）,granulocyte colony-stimulating factor（G-CSF）were used to induce NETs;NETs inhibitor DNase I or anti-CXCR1/2 antibodies were added to some groups.NETs were detected by immunofluorescence.Human neutrophils were co-cultured with four GC cells（BGC823,SGC7901,AGS and MKN45）by transwell chamber assays,and above agonists were added to generate NETs,DNase I or anti-CXCR1/2 were added to some groups.Western blotting and q RT-PCR showed the expression of BRF1 in GC cells.NE-DNA complexes in peripheral blood of mice were measured by ELISA,which was used to reflect the quantification of NETs.Cell Counting Kit-8（CCK-8）assay and colony formation assay were used to explore the proliferation of GC cells in each treatment group.The invasion and migration of GC cells in each treatment group were observed by Transwell assay.The si RNA knockdown technologies were used to silence BRF1 expression in GC cells;The CCK-8 and colony formation assay were conducted to explore the effect of BRF1 on cell proliferation in vitro;After direct co-cultured with each neutrophils treated group,the expression level of NETs was detected by immunofluorescence.BRF1-overexpression GC cells were constructed and NETosis of the co-cultured neutrophils were detected by immunofluorescence.Mouse Forestomach Carcinoma（MFC）was selected for the study,and the peripheral blood neutrophils of mice were derived from BALB/c mice.The co-culture system construction of peripheral blood neutrophils with GC cells and cell function methods in mice were the same as those of human cells.Results In vitro,neutrophils were isolated and induced to form NETs using IL-8 and its murine homologues,PMA and G-CSF,which was attenuated by the administration of DNase I.NETs were also produced when neutrophils isolated from murine peripheral blood stimulated by CXCL1/2（IL-8 homologues）,which were destructed by murine CXCL1-neutralizing antibody or/and murine CXCL2-neutralizing antibody.Extensive NETs were also observed when IL-8,PMA,G-CSF-stimulated neutrophils co-cultured with GC cells.NETs promoted the proliferation,migration and invasion of GC cells,which were abolished by the administration of DNase I.Tumorigenesis was decreased in the DNase I treated mice as demonstrated by significant growth retardation and decreased NETs formation in tumor tissues and reduced plasma NE-DNA levels.CXCR1/2 inhibition ameliorated the NETs induced proliferative effect of GC in vitro and CXCR2 inhibition in vivo attenuated the progression of GC,as indicated by significant growth retardation and decreased NETs formation in tumor tissues and reduced plasma NE-DNA levels.NETs promote tumor progression through affecting the expression of BRF1 and p21/p27 in GC cells and regulating BRF1 of GC cells can influence NETosis of the co-cultured neutrophils and itself proliferation.Conclusion NETs induced by IL-8-CXCR1/2 provide a microenvironment conducive to tumorigenesis through influencing BRF1 and cyclin p21/p27 of GC cells.DNase and anti-CXCR2 administrations may be novel therapeutic interventions in GC patients.