Identification And Characterization Of The Roles Of CircCASP9 In Gastric Cancer Based On A CircRNA–miRNA–mRNA Regulatory Network

Background:Gastric cancer(GC)is a malignant tumor with high morbidity and mortality worldwide.The incidenceof GC is mainly attributed to a variety of pathogenic infections,including Helicobacter pylori and EB virus.At present,the treatment methods for GC patients include surgery,chemotherapy and targeted therapy.The 5-year survival rate of patients with stage IA and IB GC is 60%-80%,while the 5-year survival rate of patients with stage III GC is only 18%-50%.The main reasons for low survival rate of GC are tumor metastasis,tumor heterogeneity,and chemoradiotherapy resistance.Therefore,exploring new biomarkers and therapeutic targets for GC is crucial.Recently,numbers of non-coding RNAs have been found to be closely related to the occurrenceand development of GC.Circ RNAs are a new class of endogenous non-coding RNAs,formed by covalently binding 5’ and 3’ ends and generated by reverse splicing of introns or exons.Increasing evidences have confirmed that circRNA affect the progression of various malignant tumors,and can be used to monitor the occurrenceand prognosis of tumors,which has the potential to become a new tumor biomarker and therapeutic target.There are three main mechanisms of cir RNAs :(1)competitively binding miRNA to inhibit the degradation of target genes,thus regulating the biological behavior of cells;(2)binding with RNA binding proteins(RBP),affecting protein function or regulating downstream target gene level;(3)circRNA with open reading frame(ORF)and internal ribosome entry sites(IRES)can be translated into short peptides that play biological roles and affect the occurrenceand development of tumors.In this study,we found and identified a new circRNA(hsa＿circ＿0010039)involved in GC progression.Hsa＿circ＿0010039 is a circRNA with significant differential expression screened out from circRNA microarrays(Note: derived from chr1:15844604-15844890,it is composed of exon 10 of the parent gene CASP9,which is reverse cut into a ring,with a size of 286 bp,and named circCASP9).We found that the expression level of circCASP9 in GC tissue was significantly reduced.Circ CASP9 significantly inhibited the proliferation and metastasis of GC cells in vitro and in vivo.In order to identify the candidate target gene of circCASP9,a competitive endogenous RNA(ceRNA)network was constructed with one circCASP9,2 miRNAs and 55 m RNAs by bioinformatic analysis.Finally,we verified that circCASP9 can be used as a sponge of miR-589-5p to regulate the expression of KANK1 to regulate the progress of GC.These results suggest that circCASP9 may be a potential biomarker and therapeutic target of GC.Objective:1.To clarify the biological function of circCASP9 in GC;2.To establish a new circRNA-miRNA-m RNA ceRNA network;3.To prove the regulation effect of circCASP9/miR-589-5P /KANK1 axis on proliferation,migration and invasion of GC.Methods:1.The differential expression circRNAs were obtaiend from our circRNA microarray and the GC circRNA microarrays downloaded from GEO.q RT-PCR was used to detect the expressions of circRNA in GC tissues and adjacent normal tissues.Furthermore,the expression of circCASP9 was detected in a series of GC cell lines and normal gastric epithelial cell lines.FISH array was used to detect the localization of circCASP9 in human GC cells.The expression of circCASP9 in GC tissues were used to analysis the correlation with the clinical data of these patients.2.GC cell lines with overexpression or interferenceof circCASP9 were constructed.CCK-8,Ed U,Colony Formation and Transwell assays were used to detect the effects of circCASP9 in GC cells on proliferation,migration and invasion.GC cell lines which overexpressed circCASP9 were used to construct subcutaneous tumorigenesis and liver metastasis models in nude mice.Immunohistochemistry(IHC)was used to detect the expression level of Ki-67 in nude mouse tumor.3.The downstream miRNAs of circRNA and the downstream m RNAs of miRNA were obtained by bioinformatic analysis.The ceRNA network of circRNA/miRNA/m RNA was constructed by the Cytoscape software.The competitive binding relationship between circRNA and miRNA was verified by dual-luciferase reporter system and FISH assay.Literature analysis identified the GC suppressor m RNA in ceRNA network.m RNA was screened out because western bolt and q RT-PCR verified its expression level was affected by circRNA and consisted with the expression of circRNA.The competitive binding relationship between miRNA and m RNA was verified by dual-luciferase reporter system.q RT-PCR was performed to verify whether the regulation of circRNA on m RNA could be reversed by miRNA.Salvage experiments was conducted to verify whether the effects of circRNA on GC proliferation,migration and invasion could be eliminated by miRNA.Results:1.The expression of circCASP9 was low in human GC tissues and a series of GC cell lines.FISH assay showed that circCASP9 was located in cytoplasm and nucleus.The expression of circCASP9 was negative correlated with the tumor size and the number of lymph metastasis.2.In vitro experiments,circCASP9 could inhibit proliferation,migration and invasion ability of GC cells.In vivo experiments,circCASP9 could inhibit subcutaneous tumorigenesis and liver metastasis in nude mice.IHC showed that the expression of Ki-67 in nude micetumor was decreased in the circCASP9 over-expressed group.3.A new circRNA-miRNA-m RNA ceRNA network of GC was constructed by Cytoscape.Circ CASP9 and miR-589-5p have competitive binding relationship in GC cell lines identified by dual-luciferase reporter system and FISH.Western blot and q RT-PCR verified the the expression of KANK1 was affected by circCASP9 and consisted with the expression of circCASP9.Mi R-589-5p and KANK1 have competitive binding relationship in GC cell lines identified by dual-luciferase reporter system.q RT-PCR verified the regulation of circCASP9 on KANK1 could be reversed by miR-589-5p.Salvage experiments verified the effects of circCASP9 on GC proliferation,migration,invasion and the expression of KANK1 could be eliminated by miR-589-5p.Conclusions:Circ CASP9 inhibited the proliferation,migration and invasion of GC by miR-589-5p/KANK1 axis.