EIF4G1 Promotes Gastric Cancer Progression By Regulating DKK1-AKT Signaling

Gastric cancer is one of the most common and fatal tumor.The causes of gastric cancer are more complicated,such as Helicobacter pylori infection,EB virus and some gene mutations.The treatment of gastric cancer often adopts conventional treatment methods such as surgery,radiotherapy and chemotherapy,but most of the patients are already in the middle and late stages when they are diagnosed,resulting in an unsatisfactory 5-year survival rate.With the rapid development of translational medicine,there is an urgent need to discover some new tumor markers or therapeutic targets to facilitate the early diagnosis and treatment of gastric cancer.Eukaryotic translation initiation factor 4G1,a member of the EIF4 F family,is involved in the regulation of cap-dependent and cap-independent translation initiation.It has been reported that EIF4G1 is closely related to the occurrence and development of various cancers.We used the TCGA database analysis to find that EIF4G1 is highly expressed in the tumor tissue of gastric cancer patients.However,whether EIF4G1 is involved in regulating the occurrence and development of gastric cancer and its specific regulatory mechanism are still unclear.Therefore,this study mainly focused on EIF4G1,obtained gastric cancer cell lines with stable knockdown of EIF4G1 through lentiviral packaging system,and explored its specific role and mechanism in the occurrence and development of gastric cancer by in vitro and in vivo experiments so as to provide new ideas for the treatment of gastric cancer.Part 1.Correlation between EIF4G1 expression and clinicopathological characteristicsAims: To clarify the expression level of EIF4G1 in gastric cancer patient tissues and analyze the correlation between the expression of EIF4G1 and the clinical information of patients.Methods:1)Using the TCGA database to analyze the expression of EIF4G1 in gastric cancer tissue and normal tissue2)The Kaplan-Meier Plotter database was used to analyze the effect of EIF4G1 expression on the prognosis of gastric cancer patients.3)To detect the expression of EIF4G1 in gastric cancer patient tissues and corresponding adjacent tissues by immunohistochemistry,and to analyze the correlation between EIF4G1 and the clinical information of gastric cancer patients.4)The expression of EIF4G1 in gastric epithelial cells and gastric cancer cells was detected by western blotting.Results:1)The expression level of EIF4G1 in gastric cancer tissues was higher than that in normal tissues.2)The overall survival,first progression,and post progression survival of gastric cancer patients with high EIF4G1 expression were significantly lower than those of gastric cancer patients with low EIF4G1 expression.3)Immunohistochemical analysis of tissue microarray showed that the expression of EIF4G1 in gastric cancer tissue was significantly higher than that in the corresponding adjacent tissue,and the expression level of EIF4G1 was positively correlated with the pathological grade of gastric cancer patient tissue.4)The expression of EIF4G1 in gastric cancer cells was relatively higher than that in gastric epithelial cells.Conclusion: EIF4G1 was highly expressed in gastric cancer tissues,and the expression of EIF4G1 was correlated with the clinical information of gastric cancer patients,including affecting the survival time of gastric cancer patients,and was correlated with the histopathological grade of gastric cancer patients,suggesting that EIF4G1 could be involved in regulating the occurrence and development of gastric cancer.Part 2.The role of EIF4G1 in gastric cancer progressionAims: To figure out the functions of EIF4G1 in regulating gastric cancer cells in vivo and in vitro.Methods:1)Using the lentiviral packaging system,MGC-803 and SGC-7901 cells were infected with the virus and selected with puromycin for 7 days to establish stable cell line,and the knockdown efficiency was detected by Western blot.2)In cells with stable knockdown of EIF4G1 and control cells,the effect of EIF4G1 on the proliferation of gastric cancer cells was detected by tumor-bearing model and CCK8 experiments.3)Colony formation analysis of control and EIF4G1 knockdown in gastric cancer cells.4)Using wound healing test to detect whether EIF4G1 affects the migration ability of gastric cancer cells.5)Transwell assay was used to detect the effect of EIF4G1 on the invasion ability of gastric cancer cells.Results:1)Western blot analyses were performed to verify that sh RNA targeting EIF4G1 can effectively downregulate the expression level of EIF4G1.2)Knockdown of EIF4G1 could effectively inhibit the proliferation ability of gastric cancer cells in vitro and inhibit tumor size in vivo.3)Knockdown of EIF4G1 could significantly inhibit the colony formation of gastric cancer cells.4)Silencing of EIF4G1 inhibited the malignant behavior of gastric cancer cells,including the migration and invasion ability of gastric cancer cells.Conclusion:We confirmed that EIF4G1 can participate in regulating the growth,proliferation,migration and invasion of gastric cancer cells in vitro and affecting tumor size in vivo,suggesting that EIF4G1 can act as an oncogene to promote the occurrence and development of gastric cancer.Part 3.The mechanism of EIF4G1 promoting gastric cancer progression through DKK1-AKT signaling pathwayAims: To reveal the mechanism of EIF4G1 regulating gastric cancer progression.Methods:1)Differential protein screening was obtained by knocking down EIF4G1 in gastric cancer cells and gastric epithelial cells,using high-throughput protein microarrays(containing 440 protein markers),and using KEGG and GO enrichment analysis to obtain signaling pathways and functions regulated by EIF4G1.2)Using Venn analysis,we identified that DKK1 is a downstream substrate of EIF4G1 in gastric cancer cells but not in gastric epithelial cells.3)The TCGA database was used to analyze the expression of DKK1 in gastric cancer tissue and its correlation with the prognosis of gastric cancer patients.4)Western blot and q-PCR verify that EIF4G1 regulates the expression of DKK1.5)To detect whether DKK1 gene affects the biological function of gastric cancer cells,and whether the effect of EIF4G1 on the biological function of gastric cancer cells depends on DKK1.6)Western blot detected whether EIF4G1 and DKK1 can promote the activation of AKT,and further detected whether the activation of EIF4G1 to AKT depends on DKK1.7)The cells were treated with AKT inhibitor MK2206 to detect whether the effect of EIF4G1 on the biological function of gastric cancer cells depended on the activation of this signaling pathway.8)To determine the relevance of regulation of DKK1-AKT signaling by EIF4G1 in patients,we performed immunohistochemical staining of p-AKT,EIF4G1,and DKK1 on the gastric cancer tissue microarrays.Results:1)The KEGG and GO enrichment analysis results of the differential proteins obtained by the protein chip confirmed that EIF4G1 can participate in the regulation of MAPK,PI3K-AKT and other signaling pathways and the regulation of cell motility.2)The results of Venn analysis showed that DKK1 was the downstream target protein of EIF4G1.3)TCGA data showed that DKK1 was overexpressed in gastric cancer tissues,and the patients with high DKK1 expression had poor survival.4)Western blot and q-PCR confirmed that knockdown of EIF4G1 significantly down-regulated the expression of DKK1.5)Using transwell experiments,it was confirmed that knockdown of DKK1 can significantly inhibit the migration and invasion ability of gastric cancer cells,and DKK1 mediates EIF4G1 to regulate the migration and invasion of gastric cancer cells.6)Western blot analysis confirmed that overexpression of EIF4G1 leads to the upregulation of DKK1 expression and promotes AKT activation,and EIF4G1 regulates AKT signaling pathway dependent on DKK1.7)Through functional experiments,adding AKT protein inhibitor MK2206 to cells,EIF4G1 could not regulate the migration and invasion of gastric cancer cells,suggesting that the effect of EIF4G1 on the migration and invasion of gastric cancer cells depends on the AKT signaling pathway.8)EIF4G1 expression is positively correlated with DKK1 expression and with AKT phosphorylation on Ser473 in clinical gastric cancer samples.Conclusion:First,DKK1 was identified as a downstream target protein of EIF4G1,and knockdown of EIF4G1 significantly inhibited the expression of DKK1.Silencing of DKK1 can significantly inhibit cell migration and invasion,and DKK1 mediates the regulation of EIF4G1 on gastric cancer cell migration and invasion.Both EIF4G1 and DKK1 can promote the activation of AKT,thereby activating the AKT signaling pathway,and EIF4G1 regulates the activation of AKT through DKK1.Finally,it was confirmed that the regulation of EIF4G1 on gastric cancer cell migration and invasion depends on the AKT signaling pathway,and EIF4 G was positively correlated with the expression of DKK1 and P-AKT(S473)proteins in gastric cancer tissue samples.Therefore,this part revealed a new mechanism by which EIF4G1 promotes the migration and invasion of gastric cancer cells through the DKK1-AKT signaling pathway.