| Objective: To investigate the role of Helicobacter pylori(H.pylori or HP)infection in the activation of normal fibroblasts(NFs)into cancer-associated fibroblasts(CAFs),and explore the interaction of CAFs with gastric cancer cells and its molecular mechanism.Methods:(1)NFs,CAFs and AGS cells were infected with H.pylori 1004 for 7 days at a multiplicity of infection(MOI)of 50,and the growth of H.pylori 1004 and urease B protein expression were subsequently detected by immunofluorescence(IF)and Western blot,respectively.(2)NFs activation experiments were divided into direct infection and indirect infection experiments.Direct infection was divided into 3 groups:(1)NFs were directly infected with H.pylori 1004 for 7 d,(2)NFs and AGS cells were co-cultured for 7 d.and(3)H.pylori 1004,NFs and AGS cells were co-incubated for 7 days.Indirect infection experiments were also divided into 3 groups:(1)The upper of Transwell chamber with 0.4 ?m pore size was H.pylori 1004 and the lower was NFs,which were co-cultured for 7 days,(2)The upper was AGS and the lower was NFs,which were co-cultured for 7 days,and(3)The upper was the infected AGS cells with H.pylori 1004,and the lower was NFs cells,which were cultured for 7 days.Western blot was used to detect the expression of Fibroblast-activating protein(FAP)and Lumican(LUM)proteins in NFs to evaluate the activation of NFs.Finally,Transwell assay was used to detect the effects of NFs activation on the cell migration and invasion of AGS.(3)Interaction experiments between CAFs and AGS were conducted by using Transwell chamber with 8 ?m pore size and were divided into 4 groups:(1)AGS(the upper of Transwell)/ CAF(the lower of Transwell)group as a control.(2)AGS(the upper of Transwell)/ CAF+HP1004(the lower of Transwell)group,in which CAF was infected with H.pylori 1004 for 7 days.(3)AGS+HP1004(the upper of Transwell)/ CAF(the lower of Transwell)group,in which AGS cells were infected for 7 days.(4)AGS+HP1004(the upper of Transwell)/ CAF+HP1004(the lower of Transwell)group,in which AGS and CAF cells were simultaneously infected with H.pylori 1004 for 7 days.All of cells were cultured for 24 h(migration)and 72 h(invasion),respectively,later,AGS cells migrated or invaded into lower chamber of Transwell were counted by crystal violet staining.(4)The medium in the NFs activation experiment and the interaction experiments between CAFs and AGS were collected and detected by cytokine chip.Finally,interleukin-8(IL-8)and Serpin family E member 1(Serpin E1)concentration in medium was detected by ELISA.(5)The relationship between Serpin E1 and gastric cancer was analyzed using the Human Protein Atlas(HPA)database and the GEPIA online tool.(6)Three-dimensional culture model was constructed with Matrigel and type I rat tail collagen.CAFs or NFs and gastric cancer SGC-7901 cells were inoculated on the gel for 10 days,then the matrigel was removed,fixed by formalin,embedded by paraffin and subjected to HE staining.Results:(1)The results showed that after 7 days infection of NFs,CAFs and AGS with H.pylori 1004,H.pylori 1004 could still adhere to cells and grow,and express the characteristic protein Urease B.(2)The results of NFs activation experiments:(1)Whether direct or indirect infection,FAP and LUM expression in NFs were significantly enhanced in H.pylori 1004 and NFs co-cultured group,AGS and NFs co-cultured group and H.pylori 1004 AGS and NFs co-cultured group(P <0.05),and NFs infected with H.pylori 1004 could promote the migration and invasion of AGS cells(P <0.05),suggesting that both H.pylori infection and AGS can promoting the activation of NFs.(2)The results of cytokine microarray showed that after NFs were infected with H.pylori 1004,the levels of cytokine CCL2,Serpin E1,IL-6 and CXCL12 increased significantly.When AGS was co-cultured with NFs,10 cytokines included IL-8 and IL-6 in culture medium increased.The results suggested that these cytokines are related to he activation of NFs induced by H.pylori and AGS.(3)The levels of Serpin E1 and IL-8 in medium were significantly increased(P<0.001)and decreased(P <0.01),respectively,after NFs and AGS were infected with H.pylori 1004 or NFs,AGS and H.pylori 1004 were co-incubated.(3)The results of the interaction experiments between CAFs and AGS:(1)The infection of CAFs or AGS cells with H.pylori 1004 alone can promote AGS cell migration and invasion,but when CAFs and AGS were simultaneously infected with H.pylori 1004,the promoting effect of migration and invasion were strongest(P<0.05).(2)Cytokine chip results showed that Serpin E1 levels increased significantly when AGS cells were infected with H.pylori 1004,and CXCL1,CXCL10 and CCL5 content significant increased when CAFs or both CAFs and AGS cells were infected with H.pylori 1004,suggesting that these cytokines are involved in the interaction between CAF and AGS.ELISA results also consistently confirmed that Serpin E1 and IL-8 levels in medium were increased significantly.(3)Data analysis showed that the expression of Serpin E1 in gastric cancer tissues was higher than peritumoral tissues,and it was mainly expressed in the fibroblasts of gastric cancer tissues,and its high expression was negatively correlated with the 10-year survival rate of patients.Conclusion:(1)Both H.pylori infection and co-culture of NFs with AGS cell can promote the activation of NFs,and when the two of them directly treat NFs,the activation effect is the strongest.(2)H.pylori infection can enhance the interaction between CAFs and AGS by promoting the migration and invasion of AGS cells.(3)H.pylori infection can induce the secretion of Serpin E1 and other cytokines,which plays an important role in the activation of NFs and the interaction between CAFs and AGS. |
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