Study Of RACK1 Gene In Regulation Of The Biological Behavior And Mechanism In Gastric Cancer

Background: Gastric cancer(GC)is the most common malignant tumor of the gastrointestinal malignancy and the third leading cause of cancer-related mortality worldwide.According to statistics,in 2008,there were 989600 new cases and 738,000 deaths worldwide of GC,with 40% of new cases occurring in China.Unfortunately,most patients with GC are diagnosed with advanced or metastatic disease in China,and chemotherapy is the primary treatment for these patients,while the overall benefits of chemotherapy are largely limited due to drug resistance.Therefore,it is of great significance to select functional genes closely related to the development of GC and provide new diagnosis targets and potential therapies for patients with GC.Receptor of activated C-kinase 1(RACK1),also known as GNB2L1,is a member of the Trp-Asp40(WD40)-repeat protein family.As an intercellular scaffold protein,RACK1 has been found to play a critical role in a wide range of biological responses,including cell growth,migration,differentiation,signal transduction,and immune response by combining with proteins associated with various signal transduction pathway.RACK1 exerts dual functions both in cell growth and apoptosis.In recent studies,RACK1 is found to be involved in proliferation,invasion and migration process of tumor cells,and presents an organic-specific expression in different tumors.Inconsistent RACK1 expression has been found in various primary cancer tissues,as several studies have shown that RACK1 is frequently upregulated as an oncogene in lung cancer,breast cancer,nasopharyngeal carcinoma,whereas downregulated in GC.We found RACK1 is a potential prognostic factor of solid tumors when conducting a meta-analysis and its high expression indicates poor prognosis.But only one gastric cancer study in meta-analysis,in contrast with the meta conclusion.We further found that the expression of RACK1 in gastric cancer was down regulated by public database analysis,and it was found that its expression was positively correlated with the prognosis of gastric cancer through the analysis of GEO database.At present,there are few studies on the relationship between RACK1 and gastric cancer.The mechanism of its effect on gastric cancer is not fully studied,and the study of the relationship between RACK1 and autophagy has not been reported.The purpose of this study was to detect the effect of RACK1 on the biological behavior of gastric cancer cells,and to explore the mechanism of action from the angle of autophagy.Method: 1.A systematic search of eligible studies was conducted in the PubMed,Web of Science and Cochrane Library databases as of Feb 16,2018.We aimed to explore comprehensively the potential role of the RACK1 as a prognostic marker in multiple cancers.Analyses of the pooled data were performed,and the odds ratio(OR)or hazard ratio(HR)and the 95% confidence interval(95% CI)were calculated and summarized to evaluate the strength of this association using a fixed or random-effects model.This meta-analysis was performed with STATA software version 12.0(Stata Corp LP,TX,USA).2.A totals of 30 fresh-frozen GC tissue and paired non-cancerous tissue samples were collected from the First Affiliated Hospital of China Medical University.Corresponding RACK1 RNA and proteins were extracted.RT-PCR and Western Blot methods were used to detect the expression.The significance of m RNA expression of RACK1 to prognosis in GC was analyzed by using the public databases(GEO/TCGA).3.HI assay,Transwell migration assay and invasion assay were used to evaluate the effects of RACK1 gene on migration and invasion ability of GC cell lines BGC823 and HGC27.The flow cytometry were used to analyze the effects of RACK1 on cell cycle.The clonogenic assay and MTT assay were used to detect the effect of RACK1 on proliferation activity.4.Mouse xenograft study was used to detect the change of the cells’ proliferation in vivo.Western blot was used to detect the correlative proteins of EMT signaling pathways and autophagy and AKT/m TOR signaling pathways when upregulated the expression of RACK1 in GC cell lines.Results: 1.The pooled HR in meta-analysis indicated that high RACK1 yielded worse survival in different cancers(1.69,95% CI: 1.26-2.27,p<0.001).2.The expression of RACK1 in 30 paired GC and noncancerous gastric tissues were analyzed by using q RT-PCR and Western Blot which showed that m RNA and protein expression levels of RACK1 were evidently lower in GC tissues than in adjacent noncancerous tissues(P<0.05).The prognostic value of RACK1 expression in 876 patients with GC was assessed by the public databases(GEO/TCGA).Kaplan–Meier analysis showed that high expression of RACK1 was strongly associated with better overall survival.3.The abilities of proliferation and the migration and invasion activity of the GC cells were decreased significantly by upregulating the expression of RACK1 by plasmid transfection in cell lines BGC823 and HGC27,GC cells cycle arrested in G0/G1 phase.4.The transplanted tumor of BGC-823 cells in nude mice decreased,the tumor weight and volume were lower than the control group(P<0.05).Western Blot showed the protein expressions of E-cadherin was upregulated and of Vimentin was downregulated in cells that RACK1 was overexpressed.An apparent increase in the levels of autophagy associated protein LC3B-II and Beclin1 in BGC-823 and HGC-27 RACK1 overexpression cells was detected while a decline in that of p62,p-AKT and p-m TOR.Conclusion: 1.The expression of RACK1 is lower in tumor tissues.The expression of RACK1 is associated with the prognosis of GC patients.High expression of RACK1 shows better prognosis.2.Overexpression of RACK1 suppresses proliferation,migration and invasion of GC cell lines BGC823 and HGC27 by affecting EMT signaling pathway.3.Overexpression of RACK1 induces autophagy in BGC-823 and HGC-27 cells by inhibiting AKT/m TOR signaling pathway.4.RACK1 may be a potential prognostic marker in solid cancers.