Study On The Role And Mechanism Of ARFGEF3 In Proliferation And Metastasis Of Gastric Cancer

Gastric carcinoma(GC)is a common malignant tumor in the digestive system which derived from epithelial cells.In China,gastric carcinoma ranks third in malignant tumor morbidity and mortality and threat the public health.Due to most of the early gastric carcinoma patients has no obvious clinical symptoms,and lack of specific tumor marker,most of the gastric carcinoma patients were diagnosed as middle or advanced stage at initial diagnosis.Tumor invasion and metastasis have been existed in above patients and five years survival rate of these patients is no more than 30%.Hence,further exploring the molecular mechanisms of gastric carcinoma development is necessary to find tumor marker or potential therapeutic target,which plays an important role in gastric cancer diagnosis and treatment.ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor 3(ARFGEF3)is a newly discovered member of ARFGEFs family.The latest literature revealed that ARFGEF3 is abnormally elevated in some tumors,which is correlated with malignant biological behavior and worse prognosis of tumors.However,the distribution and expression of ARFGEF3 is still unclear in human normal tissues and malignant tumor tissues.Does abnormal expression of ARFGEF3 exist in gastric carcinoma tissue and participate in gastric carcinoma development and progression? What role does ARFGEF3 play in this process? What is the molecular mechanism? The above questions have never been reported in the literature,Therefore,this study aims to resolve these questions.Objective:1.To investigate the expression and distribution of ARFGEF3 in human common tumors and normal tissues.2.To determine the expression of ARFGEF3 in gastric carcinoma and its relationship with clinicopathological features and prognosis.3.To clarify the role of ARFGEF3 in the process of gastric carcinoma proliferation,migration and metastasis,as well as its effect on epithelial-mesenchymal transition(EMT).4.To elucidate the potential regulatory mechanism of ARFGEF3 in gastric carcinoma cell proliferation,invasion,migration and EMT process.Methods:TIMER and GEPIA database were used to analyze the expression of ARFGEF3 in m RNA level in normal tissues and malignant tumor tissues.The ARFGEF3 protein in 23 normal tissues and 34 common malignant tumor tissues was detected by tissue microarray and immunohistochemistry(IHC).To detect the ARFGEF3 m RNA expression in gastric carcinoma tissues using TCGA database by UALCAN website.And IHC was preformed to validate the expression of ARFGEF3 protein in in 70 early gastric carcinoma(EGC)tissues,154 advanced gastric carcinoma(AGC)tissues and their paired adjacent non-tumorous tissues.The correlation of clinical features and prognosis was further analyzed according to the expression intensity of ARFGEF3 protein.The expression of ARFGEF3 m RNA and protein in human normal gastric epithelial cells and human gastric carcinoma cell lines were tested by quantitative Real-time PCR(RT-q PCR)and Western blotting(WB).ARFGEF3 sh RNA was used to silence the expression of ARFGEF3,and transfected into human gastric carcinoma cell lines with high expression of ARFGEF3.CCK8 and colony formation assay were used to detect the effects of ARFGEF3 interference on cells proliferation and clone formation of gastric carcinoma cells.And flow cytometry was used to analyze the cell apoptosis and cell cycle progression of gastric carcinoma cells after interference with ARFGEF3.Western blotting was used to evaluate the effect of protein on the critical regulatory proteins that promote cancer proliferation.In vivo,the xenografted tumor model was used to investigate the function of ARFGEF3 in gastric carcinoma growth.Scratches wound healing assay and transwell assay were used to analyze the effect of ARFGEF3 interference on the invasion and migration.Western blotting was used to examine the expression of epithelial mesenchymal transition markers,such as N-cadherin,E-cadherin,and vimentin.The expression of EMT-related marker was evaluated in xenografted tumor tissues of nude mice via IHC.Western blotting was used to detect the activities of MAPK,NF-κB and Akt /m TOR pathways in gastric carcinoma cells after knockdown of ARFGEF3.A small molecule pathway activators was added to the GC cells with ARFGEF3 knowdown,CCK8,colony formation assay and transwell invasion assay were used to detect the proliferation and migration of gastric carcinoma cells.IHC was used to analyze the correlation between ARFGEF3 and p-Akt in human GC tissues.Results1.The distribution and expression of ARFGEF3 in human normal tissues and human common tumors tissues.The expression level of ARFGEF3 m RNA and protein in breast invasive ductal carcinoma and gastric carcinoma are higher than the corresponding normal tissues.2.The expression of ARFGEF3 is elevated in GC and correlated with prognosisTissue microarray revealed that the expression of ARFGEF3 is overexpressed in gastric carcinoma tissue.In early gastric carcinoma,the expression of ARFGEF3 is related to tumor size and lymph mode metastasis.The high level of ARFGEF3 expression in advanced gastric carcinoma was significantly associated with tumor invasion depth,lymph node metastasis,TNM stage and vessel metastasis.Kaplan-Meier survival analysis exhibited AGC patients with higher expression of ARFGEF3 had a worse overall survival(OS)than patients with low expression of ARFGEF3.Univariate and multivariate analysis further showed that ARFGEF3 protein level were independent prognostic factors in patients with AGC.3.ARFGEF3 promotes proliferation and inhibits apoptosis of GC cellsCCK-8 assay and clone formation showed that the proliferation number of AGS and MKN-45 is lower than negative control group after the knockdown of ARFGEF3.Flow cytometry results showed that ARFGEF3 knockdown increased apoptosis and led to obvious G1/S phase transition arrest.Furthermore,the expression levels of cell proliferation essential protein such as Cyclin D1,CDK4 and c-Myc were reduced after down-expression of ARFGEF3.Tumor xenograft studies indicated that ARFGEF3 knockdown significantly inhibited the volumes and weight of the tumors,and cellular proliferation marker Ki-67 expression was decreased in silencing ARFGEF3 tumor tissues.4.ARFGEF3 promotes gastric carcinoma cells invasion and metastasisScratch wound healing and transwell assay showed that the invasion and metastasis of AGS and MKN-45 was greatly inhibited in ARFGEF3 knockdown group.The data from WB analyses showed that knockdown ARFGEF3 led to inhibition of EMT.The expression levels of E-cadherin were increased while N-cadherin and Vimentin were decreased in both AGS and MKN45 cells silenced by sh ARFGEF3 transfection.In vivo experiment,the expression of E-cadherin in transplanted tumor tissue is increased by ARFGEF3 knockdown,and N-cadherin and Vimentin in transplanted tumor tissue is lowly expressed.5.The mechanism of ARFGEF3 regulated biological behavior of GC cellsWB analysis result showed that there were no significant changes in the activity of MAPK and NF-κB pathways in GC cells after ARFGEF3 knockdown.However,the expression of p-Akt and p-m TOR is lowly expressed.After we used the activator of Akt in ARFGEF3 knockdown group,CCK-8 and transwell assay demonstrated a recover of gastric carcinoma cells proliferation and invasion ability which was initially inhibited by the knockdown of ARFGEF3.At the same time,WB suggested that p-Akt and p-m TOR is highly expressed in Akt activator group.IHC analysis showed positive correlation between ARFGEF3 and p-Akt expression in GC tissues.Conclusion1.ARFGEF3 is highly expressed in gastric carcinoma and is an independent risk factor affecting the poor prognosis of gastric carcinoma patients.2.ARFGEF3 participated in promoting gastric carcinoma cell proliferation,invasion and migration.The knockdown of ARFGEF3 inhibited the transfer from G1 stage to S stage and the epithelial-mesenchymal transition in gastric carcinoma cells.Furthermore,the apoptosis of gastric carcinoma cells was induced by the knockdown of ARFGEF3.3.ARFGEF3 promoted the development and progression of gastric carcinoma by the activation of Akt/m TOR signaling pathway.4.ARFGEF3 is closely related to the proliferation,metastasis,and poor prognosis of gastric carcinoma,suggesting that ARFGEF3 may become a potential molecular target for targeted therapy and prognosis of gastric cancer.