The Role And Mechanism Of Plasma Exosomal Hsa＿circ＿0000513 In The Diagnosis Of Gastric Cancer

Objective:To investigate the differentially expression of circ RNAs in exosomes of plasma from gastric cancer(GC)patients,chronic gastritis patients,and healthy individuals.A new circ RNA,hsa＿circ＿0000513(circ513),was identified by circ RNA microarry screening.A detection method for circ513 was established to evaluate its expression changes in exosomes of plasma exosomes from gastric cancer patients and to assess its value as a diagnostic marker for gastric cancer.To elucidate the role and mechanism of circ513 in the progression of gastric cancer,and to provide a new method and potential biological target for the early diagnosis and treatment of gastric cancer.Methods:Using circRNA microarray technology,differential expression analysis was performed on circRNAs in plasma exosomes from 6 pairs of GC patients,atrophic gastritis patients and healthy individuals,and the target molecule circ513 was screened out.Plasma was collected from GC patients,atrophic gastritis patients and healthy individuals,and exosomes were isolated and extracted.QRT-PCR(real-time quantitative reverse transcription polymerase chain reaction)and dPCR(digital PCR)were used to detect the expression of circ513 in plasma exosomes.The diagnostic efficacy of exosomal circ513 was analyzed comprehensively combined with clinicopathological data and ROC curve.The qRT-PCR assay using plasmids containing amplified fragments of circ513 was evaluated in terms of linear range,sensitivity and specificity.SiRNA and overexpressed plasmid were used to knock down or overexpress circ513 in gastric cancer cells,and changes in cell proliferation,migration and invasion functions were detected.Western Blot was used to detect changes in cell function-related proteins.Exosomes with high expression of circ513 were extracted from gastric cancer cells,and changes in cell function and protein were detected after co-cultured with gastric cancer cells.Nucleoplasmic separation and fluorescence in situ hybridization(FISH)were used to determine the localization of circ513 in gastric cancer cells.Bioinformatics analysis,RNA binding proteins immunoprecipitation(RIP)assay and dual luciferase reporter gene assay were used to verify the circ513-bound miRNA and its downstream pathways.Co-transfection experiments,Western Blot and cellular functional experiments were used to verify the regulatory mechanism of the downstream pathway of circ513.Results:Compared with healthy individuals,circ513 was highly expressed in plasma exosomes of gastric cancer patients(p<0.0001)and atrophic gastritis patients(p<0.05).Pathological correlation analysis showed that circ513 was closely related to tumor differentiation degree(p=0.03).The ROC analysis showed that the area of exosomal circ513for the diagnosis of gastric cancer was 0.715.Methodological evaluation showed that qRT-PCR detection of the circ513 had good specificity and linear correlation,with a quantification limit of 10~4copies/ml.Overexpression of circ513 could promote the proliferation,migration and invasion of gastric cancer cells and related proteins changes,while knockdown showed the opposite effect.Co-cultivation of exosomes with high expression of circ513 and gastric cancer cells enhanced the proliferation,migration and invasion ability of gastric cancer cells.Bioinformatics analysis and RIP experiments confirmed that circ513 could bind to AGO2protein.Dual luciferase reporter gene assay proved that circ513 could bound to miR-4695-5p,and transfection of miR-4695-5p mimics and inhibitor verified the anti-cancer effect of miR-4695-5p.Overexpression of circ513 and siRNA transfection showed that miR-4695-5p could reverse the pro-cancer effect of circ513.Bioinformatics analysis predicted that the target genes that miR-4695-5p could bind to,and dual luciferase reporter assays confirmed the binding of miR-4695-5p to WASF2.QRT-PCR detection of WASF2 expression in gastric cancer plasma exosomes showed a positive correlation between circ513 and WASF2expression.Cell functional experiments showed that WASF2 was a pro-cancer molecule,and transfection of circ513 and WASF2 showed that WASF2 siRNA could reverse the pro-cancer effect of overexpressed circ513.Western Blot preliminarily confirmed that WASF2 might be related to theβ-catenin pathway,and circ513 played a pro-cancer role through the miR-4695-5p/WASF2/β-catenin axis.Conclusions:The exosomal circ513 has certain clinical value in the diagnosis of gastric cancer.It can promote the proliferation,migration and invasion of gastric cancer cells through the miR-4695-5p/WASF2/β-catenin pathway.This study provides a novel molecular marker and target for the diagnosis and treatment of gastric cancer.