| Objective: Gastric cancer has high morbidity and mortality in China.Previous studies have shown that IL-17 B plays an important role in the development of other tumors.The role and mechanism of IL-17B/IL-17 RB signaling in gastric cancer and gastric cancer-related stem cells is not yet known.The purpose of this study was to reveal the regulatory and signal transduction mechanisms of IL-17B/IL-17 RB signaling in gastric cancer stem cells(GC-CSCs)and gastric cancer microenvironmental mesenchymal stem cells(GC-MSCs).Methods: The expressions of IL-17 B and IL-17 RB in gastric cancer tissues and gastric cancer cells were detected by qRT-PCR and Western blot.The tissue localization of IL-17 RB expression was analyzed by immunofluorescence.Gastric cancer tissue chips were purchased from Shanghai Changzheng Hospital and applied immunohistochemistry.Technical detection of IL-17 RB expression in gastric cancer tissues,clinical data and survival time of patients,analysis of the correlation between IL-17 RB expression and clinical parameters in gastric cancer tissue,and the impact on patient's prognosis.Construct IL-17 RB sh RNA lentiviral vector for knockdown of IL-17 RB expression in gastric cancer cell MGC-803(shIL-17 RB,control cell shGFP).Stimulation of shIL-17 RB cells and sh GFP cells by rIL-17 B and then the migration ability of the treated cells was detected by Transwell.The cell proliferation ability was analyzed by cloning assay.The differentiation ability of cells was detected by adipocyte induction test and the stem cell characteristics of cells were indirectly reflected.The markers of cancer stem cells in gastric cancer tissues were detected by qRT-PCR(Sall4).Expression of Lgr5,Oct4,Nanog,and Sox2 and analysis of the correlation between these CSCs labeled molecules and IL-17 RB expression.Western blot was used to verify the protein expression level.To further prove the downstream signaling mechanism of IL-17B/IL-17 RB,the phosphorylation levels of AKT and GSK-3? in MGC-803 cells treated with rIL-17 B were detected and ?-catenin expression was confirmed by immunofluorescence.The expression of ?-catenin and entry into the nucleus were confirmed.The activation of AKT was inhibited by PI3 K inhibitor LY294002,which demonstrate that the AKT/GSK-3?/?-catenin pathway was involved in the entire signal transduction process.The role of tumorigenicity was tested by subcutaneous in nude mice to analyze the occurrence and growth of gastric cancer cells.Isolation and cultivation of gastric cancer tissue microenvironment mesenchymal stem cells(GCMSCs)and umbilical cord mesenchymal stem cells(hucMSCs),after treatment of GCMSCs with rIL-17 B,detected the expression of stem cell-associated genes(Oct4,Nanog,and Sox2)and the major paracrine factors produced by MSCs IL-6,IL-8,TGF-? and CCL-5 expression by qRT-PCR.Western blot analyzed the expression of GAPDH,cyclin-D3,Nanog,Oct4,Sox2,p-STAT3(Thy705),t-STAT3,?-catenin,p-NF-kappaB and t-NF-kappaB was used to screen the main signaling pathway of rIL-17 B on MSC activation.Results: The expression of IL-17 RB in gastric cancer tissue was significantly higher than that in the adjacent gastric tissue,and it was closely related to the patient's survival time,the pathological grade of the tumor and the depth of lyophilization of the cancer tissue.The expression of IL-17 B and its receptor IL-17 RB in gastric cancer tissues is inconsistent.The expression of IL-17 B in gastric cancer tissues is not elevated,and there is no significant correlation with the expression of IL-17 RB.The content of-17 B was significantly higher in the serum than that of healthy people,while IL-25 did not change significantly.It was difficult to detect the expression of IL-17 B mRNA in gastric cancer cells.The knockdown of IL-17 RB in gastric cancer cells did not affect the proliferation and migration of tumor cells.But exogenous human recombinant IL-17B(rIL-17B)promotes cloning and migration of MGC-803 cells.This result indirectly indicates that IL-17 B may be not derived from autocrine.IL-17 RB is positively correlated with stem cell markers of gastric cancer cells.Activation of IL-17B/IL-17 RB signaling promotes the expression of stem cell specific genes(Sall4,Lgr5,Oct4,Nanog,and Sox2)in gastric cancer cells,and promotes the adipogenesis of MGC-803.IL-17B/IL-17 RB signaling enhances the activation of AKT/GSK3?/?-catenin pathway.The LY294002,an inhibitor of AKT,blocks the regulation of rIL-17 B on MGC-803 stem cell genes and ?-catenin entry into the nucleus.In vivo experiments show that exogenous rIL-17 B promoted the tumorigenesis and growth of SGC-7901 cells in vivo,and promoted the peritoneal metastasis of SGC-7901 cells.The supernatant of MSC pretreated by rIL-17 B promoted the proliferation and migration of tumor cells,rIL-17 B also promoted the proliferation and migration of GC-MSC and hucMSC cells and its expression of stem cell marker gene.rIL-17 B induces the up-regulation of paracrine IL-17 B in h UCMSCs and GC-MSCs themselves and promotes paracrine cytokines(IL-6,IL-8,TGF-?,and CCL-5)of hUCMSCs and GC-MSCs,which may be an important mechanism that influences tumor growth.Mechanically,rIL-17 B promotes the up-regulation of p-NF-?B,p-STAT3 and ?-catenin expression in hUCMSCs and GC-MSCs.Conclusion: IL-17B/IL-17 RB signaling promotes the migration and proliferation of gastric cancer cells in vitro and the occurrence and metastasis of tumors in vivo.This effect may be related to its regulation of gastric cancer stem cell function.Exogenous IL-17 B activates microenvironment stem cell-MSCs.The paracrine effect of cytokines and chemokines was significantly increased in MSCs,and the interstitial effects of gastric cancer progressed. |
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