Construction And Analysis Of GC-related CircRNA-miRNA-mRNA Networks And In Vivo Function And Clinical Significance Of Hsa＿circ＿0001829 In GC

Ⅰ Construction and analysis of circRNA–miRNA–mRNA networks related to gastric cancerGastric cancer(GC)is a high-recurrence-rate malignancy oncoma,which is the fifth most common cancer and the fourth major cause of cancer-related death worldwide.Circular RNAs(circRNAs)are a new class of noncoding RNAs that have gained increased attention in human cancer research.Mounting studies have shown that circRNAs are involved in the occurrence and development of cancers and have potential to become new biomarkers and therapeutic targets for cancers.However,there are still a large number of unknown circRNAs in GC,and their roles in GC also need to be further studied.In this study,six overlapped differentially expressed circRNAs(DECs)were screened from selected GC-related microarray and RNA-Seq datasets,and the six DECs were then validated by Sanger sequencing and Ribonudease R(RNase R)treatment.Subsequent reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analysis of GC samples confirmed decreased expressions of the six DECs(hsa＿circ＿0000390,hsa＿circ＿0000615,hsa＿circ＿0001438,hsa＿circ＿0002190,hsa＿circ＿0002449 and hsa＿circ＿0003120),all of which accumulated preferentially in the cytoplasm.Mi RNA binding sites and AGO2 occupation of the six circRNAs were predicted using online databases,and circRNA–miRNA interactions including the six circRNAs and 33 miRNAs were determined.Then,5320 target genes of the above 33 miRNAs and 1492 differently expressed genes(DEGs)from The Cancer Genome Atlas(TCGA)database were identified.After intersecting the miRNA target genes and the 889 downregulated DEGs,320 overlapped target genes were acquired.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that these target genes were related to two critical tumor-associated signaling pathways.A protein–protein interaction(PPI)network with the 320 target genes was constructed using STRING,and fifteen hubgenes(ATF3,BTG2,DUSP1,EGR1,FGF2,FOSB,GNAO1,GNAI1,GNAZ,GNG7,ITPR1,ITPKB,JUND,NR4A3,PRKCB)in the network were identified.Finally,bioactive chemicals(including vorinostat,trichostatin A and astemizole)based on the fifteen hubgenes were identifed as potential therapeutic agents for GC through the CMap analysis.In conclusion,we employed a combinative strategy of data mining,web experiments and computational biology to screen six novel circRNAs(hsa＿circ＿0000390,hsa＿circ＿0000615,hsa＿circ＿0001438,hsa＿circ＿0002190,hsa＿circ＿0002449 and hsa＿circ＿0003120),and circRNA-miRNA-mRNA networks were further constructed,which indicated that these six circRNAs might function as ceRNA to play important roles in GC.Additionally,three drugs(including vorinostat,trichostatin A,astemizole)obtained from CMap analysis were identified as potential therapeutic agents for GC.Our study provides a novel insight for exploration of the pathogenesis and therapy of GC from the circRNA-miRNA-mRNA network perspective.Ⅱ In vivo function and clinical significance of circRNA hsa＿circ＿0001829 in gastric cancerMany factors are involved in the occurrence of gastric cancer(GC),among which long-term exposure to chemical carcingens is one of the most important factors.N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)is a kind of respresentative environmental carcinogen,which is believed to be closely related to the occurrence of GC.Our previous studies have revealed that circRNAs play an important role in the the occurrence and development of MNNG-induced GC,in which hsa＿circ＿0001829has been proved to promote GC progression through miR-155/SMAD2 axis.However,the in vivo function and clinical significance of hsa＿circ＿0001829 in GC remains to be further studied.In this study,subcutaneous xenograft and metastasis models of GC confirmed that suppression of hsa＿circ＿0001829 expression inhibited tumor growth and agressiveness in vivo.Then,RNA in situ hybridization(ISH)technique was used to conduct staining analysis of hsa＿circ＿0001829 using tissue microarray in 98 cases of GC and 82 cases of adjacent normal tissues.The expression of hsa＿circ＿0001829 in GC tissues was markedly higher than that in adjacent normal tissues(p < 0.0001).The analysis of correlation between hsa＿circ＿0001829 level and clinicopathological factors in GC patients showed that hsa＿circ＿0001829 level was correlated with tumor size,N classification and AJCC 7th stage.Then combined wtih the follow-up data of GC patients,Kaplan-Meier method was used for survival analysis and result showed that the overall survival(OS)time of GC patients with high hsa＿circ＿0001829expression was significantly shorter than that of patients with low hsa＿circ＿0001829expression(p = 0.0015).Univariate and multivariate Cox regression analysis showed that expression of hsa＿circ＿0001829 was an independent prognostic indicator for the OS of GC patients.In conclusion,our study confirmed that hsa＿circ＿0001829 exert a tumor-promoting role in vivo;expression level of circRNA hsa＿circ＿0001829 was significantly upregulated in GC tissues,and GC patients with elevated hsa＿circ＿0001829 expression suggests a poorer prognosis.Moreover,hsa＿circ＿0001829 is an independent indicator of the prognosis of GC patients.