Figure 2.

hAMSC-CM-induces loss of function of circ-ABCB10 in HUVECs. (A) Changes in levels of circular RNAs that may be involved in angiogenesis in HUVECs were measured by RT-qPCR. circ-ABCB10 expression showed the highest increase, while linear ABCB10 remained unchanged. (B) Specific siRNAs targeting circ-ABCB10 were transfected into HUVECs, and the efficiency and expression of linear ABCB10 were measured by RT-qPCR. (C) Scratch test and (D) Transwell assays showed the effect of downregulation of circ-ABCB10 on HUVEC migration induced by hAMSC-CM. Scale bar, 100 µm. (E) Tube formation assay results showed that the effect of circ-ABCB10 downregulation on HUVEC angiogenesis was induced by hAMSC-CM. Scale bar, 200 µm. (F) RT-qPCR results showed changes in VEGFA mRNA expression levels in HUVECs induced by hAMSC-CM when circ-ABCB10 was downregulated (G) Western blotting results showed the corresponding changes in protein expression levels of VEGFA and p-ERK1/2. (H) Potential miR-29b-3p binding site in the 5' to 3' sequence of circ-ABCB10. (I) Dual-luciferase assay. HEK293T cells were co-transfected with hsa-miR-29b-3p or miR-NC and plasmid with wild-type or mutant circ-ABCB10. (J) The efficiency of transfection of miR-29b-3p. (K) The expression of VEGFA potentially targeted by miR-29b-3p in HUVEC transfected with inhibitor-NC or miR-29b-3p inhibitor was detected by western blot. ^*P<0.05 vs. control or NC, unless otherwise indicated. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; HUVECs, human umbilical vein endothelial cells; hAMSCs, human amnion-derived mesenchymal stem cells; CM, conditioned medium; VEGF, vascular endothelial growth factor; p-, phosphorylated; MSCs, mesenchymal stem cells; WT, wild-type; Mut, mutant; circ, circular RNA; NS, not significant.