| Objective: To search for the exosomal circular RNAs(circ RNAs)which differentially expressed in the serum exosomes of gastric cancer patients.To identify exosomal circ7989 from exosomal circ RNA sequencing as a major circ RNA for future research,such as its clinical application value and its role and regulatory mechanism in the progression of gastric cancer.To reveal the clinical significance of exosomal circ7989 and provide brand new biomarkers and possible targets for the future diagnosis and treatment of gastric cancer.Methods: Paired serum of gastric cancer patients and healthy controls were collected,and serum exosomes were isolated.Transmission electron microscopy,nanoparticle tracking analyzer and western blot were used to examine the characteristics of isolated serum exosomes.Exosomal circ RNA chip sequencing was performed on the serum exosomes that met the requirements to obtain the circ RNA differential expression profile.The removed cancer tissues and paired adjacent tissues of gastric cancer patients during surgery,serum exosome samples of gastric cancer patients and healthy controls were used for q RT-PCR detection to verify the sequencing results,and the ROC curve was drawn to analyze the diagnostic efficacy of exosomal circ7989.Gastric cancer cells were transfected with si RNA and overexpression plasmid of exosomal circ7989 to establish a functional research model.The effects of exosomal circ7989 on the proliferation,migration and apoptosis of gastric cancer cells were tested by CCK-8 assay,flat plate clone formation assay,transwell assay,flow cytometry and western blot.Online database was applied to predict mi RNAs that circ7989 might bind to,and a dual-luciferase reporter assay was performed to verify it.A co-transfected cell model of mi R-1200 and circ7989 was established,and the relationship between the biological functions of the two was detected by in vitro cell function experiments.The downstream regulatory signaling pathway of mi R-1200 was analyzed by bioinformatics,western blot and q RT-PCR were used to confirm that circ7989 regulated TGF-β/Smad signaling pathway by binding to mi R-1200.Results: Serum exosomes from gastric cancer patients and healthy controls were small disc-shaped vesicles with a diameter of 50-120 nm,and expressed positive markers CD63,CD81 and HSP70 on exosome membrane surface,but not negative marker calnexin.The results of serum exosomal circ RNA sequencing showed that circ7989 was significantly down-regulated in gastric cancer tissues and serum exosomes of gastric cancer patients.ROC curve analysis illustrated that the area under the curve of circ7989 for diagnosis in serum exosomes was 0.78,and the area under the curve for diagnosis in tissues was 0.644.The diagnostic performance of circ7989 in serum exosomes was better than that in tissues.Knockdown of circ7989 in gastric cancer cell lines HGC-27 and SNU-1 enhanced their proliferation,clone formation and migration,and decreased the proportion of apoptosis.Overexpression of circ7989 in AGS and MKN-28 cell lines had the opposite effect.The database prediction combined with RIP experiment found that circ7989 could bind to Ago2 protein,and the dual-luciferase reporter gene experiment further confirmed that circ7989 could bind to mi R-1200.Mi R-1200 promoted gastric cancer progression and reversed the biological effects of circ7989.The TGF-β/Smad signaling pathway was identified as a downstream regulatory pathway of mi R-1200.Conclusion: Circ7989 is lowly expressed in gastric cancer tissues,gastric cancer cells and serum exosomes,and is a potential novel diagnostic marker for gastric cancer.Our findings indicate that circ7989 exerts a tumor suppressor effect by binding to mi R-1200 and inhibiting the activity of TGF-β/Smad signaling pathway,and has the potential to become a therapeutic target for gastric cancer. |
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