| Background:Gastric cancer is the most common malignant tumor of digestive system in the world,which seriously threatens the life and physical and mental health of people all over the world.According to the latest global cancer statistics in 2020,gastric cancer ranks fifth in both morbidity and mortality among digestive system tumors[1].At present,Clinical trials of early gastric cancer mainly focus on the endoscopic resection,radiotherapy and chemotherapy,immunotherapy and molecular targeted therapy.Due to the low efficiency of early screening for gastric cancer,it means that the best surgical window is missed in most patients.The serious side effects of traditional chemotherapeutic drugs are easy to produce chemotherapeutic resistance;so many screening indicators for the immunotherapy advantaged population that the application scope is limited,Molecular targeted therapy has some drawbacks such as off-target effect and high price,Therefore,it is of great significance to search for molecular biomarkers and new effective therapeutic targets for early diagnosis and prognosis evaluation in gastric cancer[2-4].In recent years,many studies have revealed that G6PC plays a role of oncogenic gene in malignant processes of many kinds of tumors,such as glioblastoma,ovarian cancer,cervical cancer,etc[5-8].However,its roles in gastric cancer remain unclear..Objective:To certify the effect of G6PC gene on the proliferation and metastasis of gastric cancer cells and explore its possible molecular mechanismMaterials and methods:1.Database analysis:1)Kaplan Meier plotter database query was used to retrieve the relationship between G6PC expression and survival in patients with gastric cancer;2)Retrieve G6PC-related signal pathways through STRING database.2.In vitro experiments:1)The expression level of G6PC protein in normal gastric mucosal somatic cells and gastric cancer cell lines was detected by Protein western blot,G6PC silent stable gastric cancer cell lines HGC-7901 and BGC-823 were constructed by lentivirus transfection technology,and the silencing effect was verified by western blot.2)MTT and EDU experiments were performed to detect the effect of G6PC gene silencing on proliferation of gastric cancer cells.The effect of G6PC gene silencing on colony formation was verified by plate cloning experiment.3)The effect of G6PC gene silencing on cell migration and invasion was detected by scratch healing assay and Transwell chamber assay.4)The expression level of VEGF protein after G6PC gene silencing was detected by western blot assay,and the angiogenesis ability of human umbilical vein vascular endothelial cells HUVECs was verified by microangiogenesis assay.5)Immunofluorescence was used to detect the expression differences of EMT-related proteins e-cadherin and Vimentin in epithelial-mesenchymal transformation.6)The changes of EMT related markers and protein kinase B/Mammalian target of Rapamycin AKT/m TOR signaling pathway related proteins after G6PC gene silencing were detected by western blot assay,and the function recovery experiment was performed by adding PI3K agonist 740Y-P.3.In vivo experiment:G6PC silenced and control gastric cancer cells were inoculated into the subcutaneous skin of Balb/C nude mice respectively to construct gastric cancer transplanted tumor model,and the effect of G6PC gene silencing on the formation and growth of transplanted tumor was detected.Results:1.High expression of G6PC is associated with poor prognosis of gastric cancer patients:With the Kaplan-Meier Plotter analysis,the expression of G6PC in gastric cancer significantly affects the prognosis of patients,and high expression levels are associated with a poor prognosis.2.G6PC gene silencing inhibits gastric cancer cell proliferation:G6PC-silenced stable gastric cancer cell lines HGC-7901and BGC-823 were successfully constructed.The results of Cell proliferation assay,plate cloning assay and EDU cell proliferation assay showed that the proliferation activity,clonal formation ability and DNA replication ability of gastric cancer cells in SH-G6PC group were inhibited compared with Con group.3.G6PC gene silencing inhibited the migration and invasion ability,angiogenesis and EMT process of gastric cancer cells:Compared with Con group,the migration and invasion ability of gastric cancer cells in SH-G6PC group were significantly decreased,and the angiogenesis ability was also inhibited.Western blot showed that the expression of e-cadherin and tight junction protein ZO-1 were significantly up-regulated.The expression of interstitial marker Vimentin,transcription factor Slug protein,Matrix Metalloprotein（MMP）family MMP-9 and MMP-2 proteins were down-regulated.4.G6PC-mediated PI3K/AKT/m TOR signaling pathway inhibits the proliferation and metastasis of gastric cancer cells:Western blot results showed that the phosphorylation of AKT,m TOR and 4EBP1 was significantly inhibited after the silencing of G6PC gene.After the addition of the PI3K pathway agonist 740Y-P,the inhibitory effect of G6PC silencing on the EMT process and PI3K pathway of gastric cancer cells was significantly reversed.5.G6PC gene silencing can significantly inhibit the growth of subcutaneous transplanted tumor in nude mice of gastric cancer:The results of gastric cancer xenograft model showed that G6PC gene silencing can inhibit the formation of gastric cancer xenograft tumor and reduce the size of tumor formation.Conclusion:1.G6PC gene silencing can inhibit the malignant proliferation in and out of gastric cancer cells.2.G6PC gene silencing regulates the invasion and metastasis of gastric cancer cells by reversing EMT process and angiogenesis.3.The inhibitory effect of G6PC gene silencing on the phenotype of gastric cancer cells may be related to the inactivation of PI3K/AKT/m TOR signaling pathway. |
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