The Potential Diagnostic Value And Mechanism Of Exosomal Hsa＿circ＿0000670 In Gastric Cancer

Objective: The circRNA hsa＿circ＿0000670(circ670),which was differentially expressed in gastric cancer(GC),was screened out from GEO databases.By measuring the expression of circ670 in tissues,serums and serum exosomes of GC patients,the potential value of circ670 as a diagnostic marker was evaluated.Further,the specific molecular mechanism of exosomal circ670 in the progression of GC was revealed,providing a new molecular target for clinical treatment of GC.Methods: Combined with GEO databases of GC tissues and plasma(GSE83521 and GSE93541),the highly expressed circRNA circ670 was screened.The serum of patients with GC were collected to extract exosomes,and the size,morphology and surfacelabeled proteins of exosomes were identified by NTA,TEM and Western blot.The expression levels of circ670 in GC cells,tissues,serum and serum exosomes were detected by q RT-PCR.Combining pathological information and ROC curve,the diagnostic value of circ670 was evaluated.The location of circ670 in GC cells(HGC-27,AGS,MKN-45)and tissues were determined by FISH and nucleo-cytoplasmic separation assay.ASO and plasmid vectors were used to knock down or overexpress circ670.Moreover,exosomes and AGS were co-cultured.The effects of exosomal circ670 on the proliferation and metastasis of GC were revealed through a series of functional experiments including CCK8,flow cytometry,migration and invasion assay.The miRNA bound to circ670 and its downstream target genes were predicted though bioinformatics,and dual-luciferase reporter assay was used to verify the results.Cotransfection,q RT-PCR and Western blot were used to verify the interaction between circ670 and miRNA and to detect the downstream pathway.Results: Compared with human gastric mucosal epithelial cell(GES-1),adjacent tissues and healthy donors,the expression of circ670 was significantly higher in GC cells,tissues,serum and serum exosomes of GC patients(P<0.01).Combined with clinicopathological parameters,the level of circ670 was related to invasion depth(P=0.0037)and TNM staging(P=0.0375).Circ670 in exosomes showed better diagnostic ability than tissues and serum(The area under the ROC curve was 0.6828).FISH assay suggested that circ670 was present in the cytoplasm of tissues,but showed different localization in each GC cell,mainly in the cytoplasm of MKN-45 and AGS,while was present in both nucleus and cytoplasm of HGC-27.After circ670 knockdown in HGC-27 and MKN-45,the proliferation and migration ability of GC cells were decreased,and the cell cycle was arrested.Western blot analysis of related proteins showed that the stem cell related indicators such as Lin28 and Sox2 were decreased.However,circ670 overexpression in AGS showed the opposite results.After co-culture of exosomes with AGS which relatively lower expressed of circ670,the proliferation,migration and stemness of cells were increased.Bioinformatics analysis showed that circ670 could bind to miR-4659a/b-3p,which was confirmed by dual-luciferase reporter assay.The expression of miR-4659a/b-3p were down-regulated in GC cells.Mi R-4659a/b-3p mimics could reverse the ability to promote the growth and migration of GC after circ670 overexpression.HBEGF was shown to be a target gene of miR-4659a/b-3p,promoting the progression of GC through circ670/miR-4659a/b-3p/HBEGF signaling axis.Conclusions: Circ670 has a diagnostic value in GC,which was more advantageous in serum exosomes.Circ670 in exosomes acted as an important molecule for intercellular communication in GC,thereby promoting the proliferation,metastasis and stemness of GC.Circ670 was partially mediated by targeting miR-4659a/b-3p and HBEGF.The exosomal circ670 could be used as a new biomarker for the diagnosis of GC.