| Background: Gastric cancer is a common malignant tumor with a poor prognosis,which poses a serious threat to human health and aggravates the global burden of disease.Its early diagnosis is extremely important,but clinical-specific biomarkers are still lacking.A large number of studies in the past few decades have shown that non-coding RNAs(including mi RNA,lncRNA,and circRNA)play a vital role in the occurrence and development of human cancers.Among them,lncRNA and circRNA have formed complex with m RNA in cancer cells.Competitive endogenous RNA(ce RNA)regulatory network.It is worth noting that systematic identification of lncRNA,m RNA and circRNA is still lacking,and their complex regulatory networks in gastric cancer are unknown.Therefore,this project aims to explore differentially expressed m RNA,lncRNA,and circRNA in gastric cancer,to reveal the complex interactions between its transcripts,to try to find new targets for the diagnosis of gastric cancer,and to further anchor the core member circ FOXO3 and study its For the effects of proliferation and metastasis,analyze the relationship between downstream target genes and prognosis,and explore new biological targets for gastric cancer.Methods: Microarray analysis was used to screen the differentially expressed m RNAs,lncRNAs,and circRNAs in gastric cancer.The expression pattern of 6 randomly selected up-regulated genes in 11 matched gastric cancer samples was verified by q RT-PCR,and constructed in the STRING system.A differentially expressed m RNA-mediated proteinprotein interaction network was explored to explore the complex interactions between them.At the same time,lncRNA-mediated cis / trans regulatory networks and circRNA-mediated ce RNA networks were constructed to study their potential functions and mechanisms in gastric cancer.In addition,the public Kaplan-Meier Plotter database was used to evaluate prognostic value of differentially expressed m RNA,lncRNA,and circRNA in gastric cancer.Then circ-FOXO3 was knocked down by si RNA mediated methods,and experimental methods such as CCK-8,flow cytometry,and transwell were used to detect the effects of circ-FOXO3 knockdown on the proliferation,metastasis and invasion of gastric cancer cells.Furthermore,we detected the impact of circ-FOXO3 on cell growth in vivo using sh RNA mediated knockdown method.Finally,high-throughput sequencing technology was used to detect changes in m RNA expression in gastric cancer cells after knocking down circ FOXO3.The STRING system was used to construct a gene-mediated protein-protein interaction network that was down-regulated after knocking down circ FOXO3.The expression of 20 core genes downstream of circ FOXO3 in gastric cancer,and the public database Kaplan-Meier Plotter was used to evaluate the prognostic value of core genes downstream of circ FOXO3 in gastric cancer.Results: This study found that 922 m RNAs,2112 lncRNAs,and 2896 circRNAs were dysregulated in gastric cancer tissues,and the expressions of lncRNA lnc-GLI3-4: 1,LMF1,OLFM4,HKDCC1,hsa＿circ＿0058819,hsa＿circ＿0058830 were compared in gastric cancer.The matched adjacent tissues were significantly elevated.Health letter analysis shows that differentially expressed m RNAs in gastric cancer are involved in regulating alanine,aspartic acid,and glutamic acid metabolism;aldosterone regulates sodium reabsorption;amino sugar and nucleotide sugar metabolism;citrate cycle(TCA cycle)BMP binding;aldehyde dehydrogenase(NAD)activity;amine binding;branch chain amino acid catabolic process;venous blood vessel development.A total of 230 lncRNA-m RNA regulatory pairs were identified in the project,of which SLC9A3 and KIAA1958 were all cis-regulated by six lncRNAs.The prediction results of KEGG signalling pathways showed that the signalling pathways involved in the regulation of these lncRNAs include adhesion and connection;aldosterone regulates sodium reabsorption Axon guidance;B cell receptor signaling pathway;ARF protein signal transduction;G protein-coupled glutamate receptor binding;L-α-amino acid transmembrane transport.The constructed circRNA-mediated ce RNA network includes a total of 34 up-regulated circRNAs,33 down-regulated circRNAs,170 up-regulated m RNAs,153 down-regulated m RNAs,and 42 mi RNAs.Bioinformatics analysis shows that these differentially expressed genes and regulation Branch-chain amino acid catabolism,glycolysis / gluconeogenesis and ARF protein signaling are significantly related.KaplanMeier survival curves showed that highly expressed GPER,COL8A1,GLT25D1,EPOR,ENG,SLC5A5,OMP,NDE1,and REM1 were significantly associated with shorter patient survival times,while highly expressed NR＿073084,ENST00000565689(LOXL1-AS1,NR＿110232,ENST00000472494(HOTTIP)and ENST00000469148(PTPRG-AS1)are significantly related to the shorter overall survival time of gastric cancer patients.They both have value in assessing prognosis.Studies on the core circRNA circ FOXO3 in this project show that they may play a role in gastric cancer The effect of oncogenes is significantly higher in gastric cancer tissues than in adjacent tissues.Knocking down circ FOXO3 can significantly inhibit the ability of AGS and N87 cells to increase,metastasize and invade,and block cell cycle progression in vivo and in vitro.Use chip technology to detect knockdown of circ FOXO3 After that,the expression of m RNA in gastric cancer cells changed,and a total of 5104 differential genes were obtained.The expression level of 2378 genes was significantly up-regulated after knocking down circ FOXO3,and the expression level of 2726 genes was significantly down-regulated after knocking down circ FOXO3.Bioinformatics Predictions show that the major genes up-regulated after circ FOXO3 knockdown Intensive defense response;innate immune response;immune response;viral defense response;immune effect process;immune system process.Down-regulated expression genes after circ FOXO3 knockdown are widely involved in metabolic processes;translation;organic biosynthesis process;biosynthesis process;cell cycle And other biological processes.In the project constructed by knocking down circ FOXO3 downregulated gene-protein-protein interaction network,three cores were revealed to up-regulate expression,three cores were down-regulated to express PPI network,and its 20 circ FOXO3 downstream cores Among the genes,CDK1,MYC,BRCA1,MCM3,RFC4,MX1,ISG15,OAS1,STAT1,DDX58,IFI35,RSAD2,B2 M,DDX60,and IFI27 are significantly overexpressed in gastric cancer.Interestingly,the overexpressed BRCA1,IFI35,MCM3,DDX60 showed a significant correlation with the longer overall survival time of gastric cancer patients,while highly expressed MX1,ISG15,DDX58,IFI27,RPS28,RPS20 showed a significant negative correlation with the patient’s survival time.Conclusion: This study comprehensively analyzed the differentially expressed m RNA,lncRNA,and circRNA in gastric cancer.For the first time,a complex regulatory network was constructed to reveal the interactions between these RNAs.It was found that the imbalance of core m RNA and lncRNA and the overall survival of gastric cancer patients It is time-dependent and provides useful information for understanding the mechanism of gastric cancer progression and exploring potential treatment and prognostic targets for gastric cancer.Through further research on circ FOXO3,it was proved that it is significantly overexpressed in gastric cancer.Silencing circ FOXO3 in AGS and N87 cells can significantly reduce the proliferation,cycle,metastasis and invasion of gastric cancer cells in vivo and in vitro and it may be involved in tumor immune response and metabolism.Process-related signal pathway regulation,and found that the core downstream genes of circ FOXO3 were significantly overexpressed in gastric cancer and significantly correlated with tumor survival time.For the first time,we elucidated the proto-cancer effect of circ FOXO3 in gastric cancer and provided new ideas for identifying new biomarkers for gastric cancer. |
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