H. Pylori Induces Serpin E1 Expression By Fibroblasts To Promote Gastric Cancer Cell Growth And Peritoneal Metastasis Via P38 MAPK-VEGFa Signaling

Objective:This study is aimed at investigating the interaction between Helicobacter pylori(H.pylori)-induced expression of Serpin family E member 1(Serpin E1)in fibroblasts and gastric cancer cells,as well as the mechanism by which fibroblast-derived Serpin E1 promotes gastric cancer cell growth and peritoneal metastasis.Methods:(1)Serpin E1 expression was detected in two gastric epithelial/carcinoma cell lines(GES-1,AGS),three primary human gastric cancer cells(GC-1,GC-2,GC-3),one gastric fibroblast cell line(Hs738)and three primary human gastric cancer-associated fibroblasts(CAF-1,CAF-2,CAF-3)by western bolt analysis.(2)Gastric fibroblasts were infected with H.pylori at multiplicity of infection(MOI)50 and Serpin E1 expression was detected in fibroblasts by Western bolt and immunofluorescence(IF),and Serpin E1 levels in culture supernatants were measured by cytokine microarray(containing 36 cytokines)and enzyme-linked immunosorbent assay(ELISA).(3)To evaluate chemotaxis,a co-culture model of human umbilical vein endothelial cells(HUVECs)and fibroblasts was constructed using Transwell chambers with HUVECs added to the upper chamber and H.pylori-infected or uninfected fibroblasts and these cells-derived conditioned medium added to the lower chamber,and the number of cells migrating from HUVECs to the face of the lower chamber was measured.(4)After construction of a co-culture model of HUVECs(lower chamber)with Serpin E1 overexpression Hs738 cells(Hs738 Serpin E1,added to the upper chamber)using Transwell chambers,the expression of vascular endothelial growth factor a(VEGFa)was detected by western bolt and IF in HUVECs.(5)Hs738 cells with Serpin E1 overexpression was co-cultured directly and indirectly with HUVECs and the concentrations of VEGFa in the co-culture supernatant were measured by EILSA.(6)Four-week-old male BALB/c nude mice(n=5 per group)were co-injected subcutaneously with primary gastric cancer cells and Serpin E1-overexpressed Hs738 cells in a 4:1 ratio into the right axil of the nude mice,and the mice were killed by excessive dosage of anesthesia 28 days after injection.The volume(V=1/2 × length × width2)and weight of xenografts were mesured to evaluate tumor growth.Furthermore,the mixed cells were co-injected intraperitoneally into nude mice at the same ratio.The mice were killed by excessive dosage of anesthesia after 28 days,and then tumor nodules in the peritoneum of the nude mice were counted and removed.Tissue sections from tumor nodules were stained by HE and immunohistochemistry(IHC)for Ki67,Serpin E1,CD31,and VEGFa.(7)HUVECs were treated with exogenous recombinant protein Serpin E1(recSerpin E1).The Transwell assay was used to detecte the chemotactic effect of recSerpin E1 on HUVECs and western bolt and IF were used to detecte VEGFa expression in HUVECs.After adding Serpin E1 antibody into HUVECs,western bolt and IF were used to detecte VEGFa expression in HUVECs and tubule formation assay was used to detect tubule formation in HUVECs.(8)RecSerpin E1,Serpin E1 antibody and MAPK signaling pathway blocker(E)-Osmundacetone were used to treat alone or co-treated HUVECs,and the expression of P38,ERK1/2,JNK and its phosphorylated proteins and VEGFa were detected by Western bolt analysis.(9)Tumor nodules(n=3 per group)from peritoneal metastases in nude mice,in the GC+Hs738 Serpin E1 and GC+Hs738 NC groups,were collected for transcriptomic study.Differentially expressed genes(DEGs)with foldchange>1.2 and FDR<0.05 were screened,and GO and KEGG pathway enrichment analysis was performed.The expression and survival analysis of top15 DEGs in gastric cancer tissues were performed using GEPIA online tool and TCGA database for gastric adenocarcinoma.Results:(1)Serpin E1 was mainly expressed in gastric fibroblasts.(2)Serpin E1 expression in fibroblasts and Serpin E1 levels in culture supernatants were increased after H.pylori infection.(3)H.pylori-infected CAFs and Hs738 cells and their conditioned medium increased the migration of HUVECs from the upper to the lower Transwell(p<0.05),promoting the chemotaxis of HUVECs.(4)Serpin E1-overexpressed Hs738 cells promoted VEGFa expression and secretion in HUVECs(p<0.05).(5)The co-injection of Hs738 Serpin E1 with GC cells promoted the growth of subcutaneous transplanted tumors in nude mice,and the volume and weight of the xenograft tumors were significantly higher than those in the group co-injected with Hs738 NC and GC cells(p<0.05).The co-injection of Hs738 cells with Serpin E1 overexpression and GC cells promoted the spread of GC cells in the peritoneal cavity,and the number of tumor nodules in the peritoneal cavity was significantly higher than that in the Hs738 NC+GC group.The HE staining showed that focal necrosis was present in the abdominal tumor nodules of the three groups,but the focal necrosis was significantly reduced in the GC+Hs738 Serpin E1 and GC+Hs738 NC groups,especially in the GC+Hs738 Serpin E1 group.The IHC analysis showed that the expression of VEGFa and CD31 in the tumor nodules increased with Serpin E1 expression increasing(p<0.05).(6)Compared with the control group,both 1ng/mL and 10 ng/mL recSerpin E1 increased the number of migrating cells from the upper to lower chamber of the Transwell in HUVECs,with the strongest effect in the 1ng/mL recSerpin E1-treated group(p<0.05).VEGFa expression was significantly increased in 2 endothelial cell lines treated by 1ng/mL and 5ng/mL recSerpin E1(p<0.05).Adding Serpin E1 antibody(1μg/mL and 2μg/mL)could inhibit recSerpin E1-induced VEGFa expression and tube formation in HUVECs.When the Serpin E1 antibody concentration reached 2μg/mL,the inhibition of VEGFa expression and tube formation was most significant(p<0.05).(7)Compared to the controls,recSerpin E1 enhanced P38 MAPK phosphorylation and VEGFa expression,but had no significant effect on ERK1/2 and JNK MAPK.The addition of Serpin E1 antibody(2μg/mL)and MAPK pathway inhibitor(E)-Osmundacetone inhibited recSerpin E1-mediated P38 phosphorylation and VEGFa expression(p<0.05).(8)The transcriptomic analyses showed that the data within GC+Hs738 Serpin E1 and GC+Hs738 NC groups are relatively concentrated and have good reproducibility.One hundred and seventy-eight DEGs were screened,among which 39 genes were up-regulated and 139 genes were down-regulated.DEGs were mainly involved in biological processes such as cellular processes,biological regulation and metabolic processes,in cellular composition such as cells and cellular components,and in biological processes such as cellular processes,biological regulation and metabolic processes.KEGG pathway enrichment analysis showed that the DEGs were involved in the butyric acid metabolism,metabolic pathways and metabolism of various substances.These signaling and metabolism pathways are closely associated with tumor development and progression.(9)The expression of 7 DEGs of the Top15 DEGs(CTSS,KRT80,PDZK1IP,SEMA3C,FSTL1,SLC27A2,LOX)was significantly higher in gastric adenocarcinoma tissues than in normal tissues,and the expression of FSTL1 and LOX was negatively correlated with the 10-year survival rate of gastric cancer patients(p<0.05).Conclusions:(1)Serpin E1 is mainly expressed in gastric fibroblasts.(2)H.pylori infection induces Serpin E1 expression in fibroblasts,and Hs738 cells with Serpin E1 overexpression promotes subcutaneous growth and peritoneal metastasis of GC cells in nude mice.(3)Serpin E1 promotes GC growth via activating P38 MAPK-VEGFa signaling.