Fig. 7

Exosomal circNRIP1 regulates AKT1 expression as well as EMT in vitro and promotes metastasis in vivo. a. We determined the significantly higher expression level of circNRIP1 in plasmatic exosomes from GC patients by qRT-PCR. b. We used a transmission electron microscope (TEM) to determine the existence and morphology of exosomes purified from GC cell medium (exosome-free FBS), scale bar = 25 μm. We further confirmed the exosomes by detecting protein markers, including CD63 and CD81, by western blot. c. Red exosome signals were found in the cytoplasm of GFP-labelled tumour cells when exo-RFP GC cells were mixed with the same amount of GFP-labelled GC cells for 72 h, scale bar = 50 μm. d. We than purified exosomes and added them into GFP-labelled MKN-45 or BGC-823 GC cells. The red signal of circNRIP1 similarly appeared in the cytoplasm of GFP-labelled GC cells after 72 h, scale bar = 50 μm. e. We performed qRT-PCR and detected higher circNRIP1 expression in exosomes purified from circNRIP1-overexpressing GC cells relative to those from NC cells. f. We detected upregulated AKT1, mTOR and EMT markers in GC cells by co-culturing them with exosomes of OV circNRIP1 GC cells (OV exosomes) for 72 h via western blot. g. According to the luciferase Intensities detected in the thoracic cavity, we found that GC cells treated with OV exosomes showed higher metastasis potential. h. We harvested lung tissues for H&E staining to characterize the cancerous nodes. Cancerous node size was consistent with luciferase intensity, scale bar = 200 μm. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001