Fig. 5.

Exosomes derived from CAFs promote endothelial cell angiogenesis by modulating the circ_0084043/miR-140-3p/VEGF axis. A-D. Circular RNA circ-0084043 acts as the sponge of miR-140–3p. A.In silico predictions indicated the complementary binding site between circ_0084043 and miR-140–3p. B. The relative expression of miR-140–3p increased in transfected endothelial cells with the miR-140–3p mimic, confirming the functionality of the miR-140–3p mimic. C. A significant reduction in luciferase activity was observed when the miR-140–3p mimic was introduced, suggesting the binding affinity between miR-140–3p and circ_0084043. The results suggest that circ_0084043 effectively functions as a sponge for miR-140–3p in endothelial cells. D. SiRNA-mediated knockdown of circ_0084043 resulted in an increase in the expression levels of miR-140–3p in HUVECs, as indicated by qRT-PCR. E-I. miR-140–3p directly targets HIF-1α to suppress VEGFA. E.In silico predictions indicated the complementary binding site between miR-140–3p and the 3′-UTR of HIF-1α transcript. F. By introducing the miR-140–3p, luciferase activity decreased, indicating the binding affinity between miR-140–3p and the 3′-UTR of HIF-1α. The results confirmed the targeting of HIF-1α by miR-140–3p in endothelial cells. G. The transcript expression of VEGFA decreased when HUVECs were treated with the miR-140–3p mimic, suggesting a suppressive effect of this miRNA on VEGFA expression. H, I. Western blot analysis showed that protein levels of VEGFA decreased in endothelial cells transfected with the miR-140–3p mimic. The original bots of Fig. 5H are provided in Supplementary Fig. S9. For quantitative analysis, the density of bands was assessed using ImageJ software and represented as relative intensity (I). J. The expression of miR-140–3p was measured in endothelial cells treated with a miR-140–3p inhibitor, confirming the functionality of the inhibitor. K, L. When miR-140–3p was inhibited in HUVECs exposed to CAF Exo (100 μg/mL), there was an increase in endothelial cell migration. However, co-inhibition of miR-140–3p and circ_0084043 diminished the promoting effect of miR-140–3p suppression on endothelial cell migration. M-O. Exosomes derived from CAFs enhanced tubularization of endothelial cells through shuttling of circ_001422. M. Representative micrographs showing that when si-circ_001422 was transfected, the ability of CAF Exo-treated endothelial cells to form capillary-like tubular structures was hindered. Importantly, the suppression of angiogenesis in CAF Exo-treated HUVECs, resulting from circ_0084043 knockdown, was rescued by co-transfection of si-circ_0084043 and the miR-153–3p inhibitor. Quantitative analyses of the tube length (N) and branch (O) of endothelial cells were calculated in three randomly selected fields. P–R. Inhibiting miR-140–3p in endothelial cells treated with 100 μg/mL CAF-derived exosomes increased the expression of VEGFA at both mRNA (P) and protein (Q) levels. Results indicated that the reduction of VEGF expression in CAF Exo-treated HUVECs, resulting from circ_0084043 knockdown, was rescued by co-transfection of si-circ_0084043 and the miR-153–3p inhibitor to some extent. The original bots of Fig. 5Q are provided in Supplementary Fig. S10. For quantitative analysis, the density of bands was assessed using ImageJ software and represented as relative intensity (R). The columns represent the means of three independent experiments, with the bars indicating the standard deviation (SD). * p-value <0.05, ** p-value <0.01, *** p-value <0.001.