Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer

Figure 1 Study design. HD: Healthy donor; GC: Gastric cancer; EGC: Early gastric cancer; LAGC: Locally advanced gastric cancer; MGC: Metastatic gastric cancer; circRNA: Circular RNA; AGC: Advanced gastric cancer.

Figure 2 Identification of exosomes and bioinformatic analysis of exosomal circular RNAs. A: Exosomes were observed by transmission electron microscopy. The scale bar is 200 nm; B: Diameter and quantity of the exosomes; C: The positive markers of exosomes (CD9, TSG101, and CD81) and the negative marker (Calnexin) were tested by Western blot; D: Clustering analysis of circular RNAs (circRNAs); E: Kyoto Encyclopedia of Genes and Genomes pathway analysis of cluster 5 of differently expressed circRNAs; F: Gene Ontology enrichment analysis of cluster 5 of differently expressed circRNAs. HD: Healthy donor; GC: Gastric cancer; EGC: Early gastric cancer; LAGC: Locally advanced gastric cancer; MGC: Metastatic gastric cancer; AGC: Advanced gastric cancer; BP: Biological process; CC: Cell component; MF: Molecular function.

Figure 3 Expression levels of exosomal hsa_circ_0079439, carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 72-4, alpha-fetoprotein, and carbohydrate antigen 125 in the test cohort. A: Absolutely quantitative detection of exosomal hsa_circ_0079439 copy number in plasma between healthy donors (HDs) (n = 22) and gastric cancer (GC) patients (n = 69) by droplet digital polymerase chain reaction; B-F: Expression levels of standard serum biomarkers in HDs and GC patients, including carcinoembryonic antigen (B), carbohydrate antigen (CA)19-9 (C), CA72-4 (D), alpha-fetoprotein (E), and CA125 (F). And all the results are shown as the mean (SD). ^aP < 0.001, ^bP < 0.0001, NS: Not significant. HD: Healthy donor; GC: Gastric cancer; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; CA72-4: Carbohydrate antigen 72-4; CA 125: Carbohydrate antigen 125; AFP: Alpha-fetoprotein.

Figure 4 Expression levels of exosomal hsa_circ_0079439, carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 72-4, alpha-fetoprotein, and carbohydrate antigen 125 in the validation cohort. A: Absolutely quantitative detection of exosomal hsa_circ_0079439 copy number in plasma from healthy donors (HDs) (n = 41) and gastric cancer (GC) patients (n = 171) by droplet digital polymerase chain reaction; B-F: Expression levels of standard serum biomarkers in GC patients and HDs, including carcinoembryonic antigen (B), carbohydrate antigen (CA)19-9 (C), CA72-4 (D), alpha-fetoprotein (E), and CA125 (F). And all the results are shown as mean (SD). ^aP < 0.0001, NS: Not significant. HD: Healthy donor; GC: Gastric cancer; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; CA72-4: Carbohydrate antigen 72-4; CA 125: Carbohydrate antigen 125; AFP: Alpha-fetoprotein.

Figure 5 Diagnostic value and expression levels of exosomal hsa_circ_0079439, carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 72-4, alpha-fetoprotein, and carbohydrate antigen 125 in total participants for gastric cancer. A: Absolutely quantitative detection of exosomal hsa_circ_0079439 copy number in plasma from healthy donors (HDs) (n = 63) and gastric cancer (GC) patients (n = 240) by droplet digital polymerase chain reaction; B-F: Expression levels of standard serum biomarkers in GC patients and HDs, including carcinoembryonic antigen (B), carbohydrate antigen (CA)19-9 (C), CA72-4 (D), alpha-fetoprotein (E), and CA125 (F); G: Receiver operating characteristic curves of hsa_circ_0079439, carcinoembryonic antigen, CA19-9, CA72-4, alpha-fetoprotein, and CA125 in distinguish GC patients (n = 240) from HDs (n = 63); H: Changes of expression levels of exosomal hsa_circ_0079439 before and after treatment. And all the results are shown as the mean (SD). ^aP < 0.05, ^bP < 0.0001, NS: Not significant. HD: Healthy donor; GC: Gastric cancer; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; CA72-4: Carbohydrate antigen 72-4; CA 125: Carbohydrate antigen 125; AFP: Alpha-fetoprotein.

Figure 6 Diagnostic value and expression levels of exosomal hsa_circ_0079439, carcinoembryonic antigen, carbohydrate antigen 19-9, carbohydrate antigen 72-4, alpha-fetoprotein, and carbohydrate antigen 125 among healthy donors, early gastric cancer patients, and advanced gastric cancer patients. A: Absolutely quantitative detection of exosomal hsa_circ_0079439 copy number in plasma from early gastric cancer (EGC) patients (n = 177), advanced GC (AGC) patients (n = 63), and healthy donors (HDs) (n = 63) by droplet digital polymerase chain reaction; B-F: Expression levels of standard serum biomarkers in EGC patients, AGC patients, and HDs, including carcinoembryonic antigen (CEA) (B), carbohydrate antigen (CA)19-9 (C), CA72-4 (D), alpha-fetoprotein (E), and CA125 (F); G: Receiver operating characteristic (ROC) curves of hsa_circ_0079439, CEA, CA19-9, CA72-4, alpha-fetoprotein, and CA125 in distinguish EGC patients from HDs; H: ROC curves of hsa_circ_0079439, CEA, CA19-9, CA72-4, alpha-fetoprotein, and CA125 in distinguish AGC patients from HDs. All the results are shown as the mean (SD). ^aP < 0.05, ^bP < 0.0001, NS: Not significant. HD: Healthy donor; CEA: Carcinoembryonic antigen; CA19-9: Carbohydrate antigen 19-9; CA72-4: Carbohydrate antigen 72-4; CA 125: Carbohydrate antigen 125; AFP: Alpha-fetoprotein; EGC: Early gastric cancer; AGC: Advanced gastric cancer.