| ObjectiveGastric cancer(GC)is among the most common cancers and the third leading cause of cancer-related death worldwide.The incidence and mortality of GC in China are both higher than the world average.A substantial proportion of GC patients are asymptomatic,or else unspecified in the early stage,and the current methods for early diagnosis and treatment of GC are still limited.CircRNA has become a hot spot in tumor research in recent years.It can act as a competitive endogenousRNA to bind to miRNA and thus participate in the regulation of downstream mRNA expression.The circcRNA-miRNA-mRNA interaction networks are closely involved in the occurrence and progression of tumors,including GC.Tumor derived circRNAs can be delivered by exosomes into the tumor microenvironment(TME)and various body fluids.They not only serve as a bridge for communication between tumor cells and TME,but also are considered as potential tumor biomarkers because they are easy to detect.Many studies have explored the possible applications of circRNAs in GC diagnosis and novel therapies.In recent years,a number of high-throughput analyses have focused on the differentially expressed circRNAs(DE circRNAs)in GC tissues and body fluids,but circulating exosomal circRNAs have not been studied.Therefore,this study aims to detect DE circRNAs in the plasma exosomes of GC patients through high-throughputRNA sequencing,and then screen out circRNAs that play important roles in GC development by functional experiments in vivo and in vitro,so as to help clarify the pathogenesis of GC and provide new clues for the diagnosis and treatment of GC.Methods(1)Exo Easy Maxi Kit(Qiagen)was used to isolate exosomes from the plasma of GC patients and healthy controls(HC).Western blot and transmission electron microscopy assays were performed to identify the properties of the isolated exosomes.High-throughputRNA sequencing and bioinformatics analysis were carried out to screen differentially expressed circRNAs(DE circRNAs).The potential roles of the DE circRNAs were speculated by applying Gene ontology(GO)and Kyoto Encyclopedia of Gene and Genome(KEGG)analysis.The real-time quantitative polymerase chain reaction(RT-q PCR)assay was performed to verify the high-throughput sequencing results,and select the circRNA for further functional study.(2)The correlation between the expression level of hsa?circ?0014606 in plasma exosomes of GC patients and the clinicopathological characteristics of patients was evaluated by statistical analysis.(3)The circ Base database(http://www.circbase.org)was used to query the sequence,parental gene and chromosome location of hsa?circ?0014606.NCBI genome tools(https://www.ncbi.nlm.nih.gov/genome/)were used for genome alignment.The Circ Bank database(http://www.circbank.cn/)was used to query the coding?potential andRNA modification of hsa?circ?0014606.(4)Lentiviral vectors for overexpressing or knocking down hsa?circ?0014606were constructed.Lentiviruses were packaged and infected GC cells to establish stable cell lines.GC cell proliferation was detected by CCK8 experiment;cell apoptosis was detected by Annexin V-Alexa Fluor 488/PI staining;cell migration ability was detected by wound healing assay;cell invasion ability was detected by matrigel-coated Transwell assay.(5)THP-1 cells were stimulated with PMA and differentiate into M0 macrophages,which were co-cultured with GC cell-derived exosomes.RT-q PCR was then performed to detect M1 macrophage markers IL-12 and i Nos,and M2 markers IL-10 and Arg-1,to determine the polarization direction of the THP-1 macrophages.(6)The target miRNA of hsa?circ?0014606 and its downstream target genes were predicted by bioinformatics analysis;dual luciferase reporter assay was performed to verify their binding and interaction.(7)GC xenograft model was constructed using NCD immunodeficiency mice to determine the effects of hsa?circ?0014606 on the tumourigenicity of GC cells in vivo,and on the expression of its target miRNA and the downstream target gene.Results(1)We collected peripheral blood samples from 5 GC patients and 5 healthy donors(HC),and successfully isolated exosomes from the plasma.By high-throughputRNA sequencing,a total of 67,880 circRNAs were detected in all plasma exosomes samples,and 1,060 circRNAs differentially expressed in GC(DE circRNAs)were screened,of which 620 were up-regulated and 440 were down-regulated.(2)By applying GO and KEGG analysis,these DE circRNAs were predicted to be involved in the cell cycle,cytoskeleton organization,cell response to DNA damage,and regulation of GTPase activity,as well as phosphatidylinositol,MAPK,thyroid hormone,chemokine,and Wnt signaling pathways.(3)RT-qPCR results showed that the selected 6 up-regulated circRNAs were significantly up-regulated in GC cells and/or GC tissues;among them,hsa?circ?0014606 was the most up-regulated,and was selected for further functional studies.(4)The expression level of hsa?circ?0014606 in plasma exosomes of GC patients was positively correlated with tumor size and invasion depth.(5)Hsa?circ?0014606 is derived from YY1AP1(YY1 associated protein 1)gene,which is an exon-intron circRNA(EIciRNA)and may have the potential ability to encode protein.(6)In vitro experiments confirmed that overexpression of hsa?circ?0014606promoted GC cell proliferation,migration and invasion;knockdown of hsa?circ?0014606 increased GC cell apoptosis.(7)AGS cells derived exosomes facilitated the polarization of THP-1 cells to M2 macrophages.AGS exosomes with hsa?circ?0014606 overexpression further promoted M2 polarization,while knockdown of hsa?circ?0014606 inhibited M2 polarization.(8)miRNA-194-5p is one of the target miRNAs of hsa?circ?0014606,and its expression is down-regulated in GC tissues.Overexpression of hsa?circ?0014606 in AGS cells significantly decreased its expression.(9)Dual luciferase reporter assay confirmed that hsa?circ?0014606 could bind to miRNA-194-5P.(10)YAP1 is predicted to be the target protein of miRNA-194-5p.The dual luciferase reporter assy confirmed that YAP1 could bind to miRNA-194-5p,which promoted the degradation of its transcript.(11)Hsa?circ?0014606 promoted the tumourigenicity of AGS cells in NCD mice xenograft model,and down-regulated the expression of miRNA-194-5p and up-regulated the expression of YAP1.ConclusionsBy high-throughput RNA sequencing,a total of 1,060 differentially expressed circRNAs(DE circRNAs)were detected in plasma exosomes of GC patients,including 620 up-regulated and 440 down-regulated ones.GO and KEGG analysis suggested that these DE circRNAs were enriched in intracellular organelle and nuclear part and associated with some important biological processes and signaling pathways in GC tumorigenesis such as cell cycle,cytoskeletal organization,cell response to DNA damage,regulation of GTPase activity,and phosphatidylinositol,MAPK,thyroid hormone,chemokine,and Wnt signaling pathways.RT-q PCR confirmed the results of high-throughput sequencing,and suggested that the up-regulated circRNA in plasma exosomes was likely to come from tumor tissues and cells.Hsa?circ?0014606 was found to be up-regulated most remarkablely in GC tissues and cells.Its expression level in plasma exosomes of GC patients was positively correlated with tumor size and invasion depth.It might act as miRNA-194-5p sponge and up-regulate the expression of YAP1,thereby affecting the proliferation,apoptosis,migration and invasion of GC cells,as well as the M2 polarization of macrophages via GC cell derived exosomes,and thus plays a promoting role in GC development. |
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