Fig. 6

CircTUBGCP4 sponge miR-146b-3p to upregulate PDK2 for activating Akt signaling pathway in HUVECs. a AGO2-RIP analysis of the binding between circTBUGCP4 and AGO2 in HUVECs transfected overexpressed circTUBGCP4 plasmid and OV-NC plasmid. b MiRNA screening from circular RNA array analysis and the CIRCinteractiome website. c The efficiency of circTUBGCP4 probe by circRNA pull-down assay in HUVECs transfected the plasmid of OV-CircTUBGCP4 and OV-NC. NC probe was the negative control. d qPCR analysis of circRNA-pulled down miR-146b-3p and miR-873-5p in HUVECs transfected OV-CircTUBGCP4 plasmid. e Enrichment of miR-146b-3p pulled down by circTUBGCP4 probe compared with NC probe in HUVECs transfected OV-CircTUBGCP4 plasmid and OV-NC plasmid. f MiR-146b-3p binding sites in circTUBGCP4. g Luciferase activity of 293T transfected with circTUBGCP4-wt/mu luciferase constructs with miR-146b-3p mimic or negative control. h p-AKT (Ser 473) and AKT expression in HCT116-Exo and SW480 Exo-treated HUVECs after 48 h using western blot. i Expression of p-AKT (Ser 473) and AKT in HUVECs treated with exosomes derived from shcircTUBGCP4-01, shcircTUBGCP4-02, and shNC. j Venn diagram of potential targets of miR-146b-3p using miRDB and Targetscan. k qPCR confirmation of possible targets in HUVECs transfected with miR-146b-3p mimic and NC mimic. l Expression of PDK2 mRNA in OV-circTUBGCP4 group and OV-NC group of HUVECs detected by qPCR. m Binding verification of PDK2 mRNA and miR-146b-3p by pull-down assay using biotin-labeled miR-146b-3p in HUVEC, NC probe for miR-146b-3p was as a negative control. n The binding site of miR-146b-3p in PDK3 mRNA-3’UTR. o Luciferase activity of 293 T transfected with PDK2-3’UTR luciferase constructs with miR-146b-3p mimic or negative control. p Rescue experiment using OV-circTUBGCP4 plasmid and miR-146b-3p mimic for detecting PDK2 expression in mRNA level and protein by qPCR and western blot. GAPDH was the internal control of whole-cell lysates. Mean ± SEM. Student's t-test, *P < 0.05, 0.001 < ** P < 0.01, *** P < 0.001