Fig. 3

Circ_0001810 acted as a ceRNA of miR-1197, upregulating AK2, the target gene of miR-1197. A The miRNA predictions targeting circ_0001810 in StarBase, CircBank, and Circinteractome. B RIP assay detected the bind of circ_0001810 to AGO2 protein in HEK-293T cells. C Dual luciferase assay examined the bind of circ_0001810 to miR-1197 in SHSY-5Y cells. Mutant circ_0001810 reporter and miR-NC mimic were used as negative controls. D RNA pull-down assay examined the in vivo bind of circ_0001810 to miR-1197 in SHSY-5Y cells. Mutant circ_0001810 overexpression vector was used as negative controls. E Protein predictions targeting miR-1197 in TargetScan, StarBase, and miRBD. F Circ_0001810 upregulated the mRNA expression of different targets in SHSY-5Y cells by RT-qPCR. G Circ_0001810 upregulated the protein expression of AK2 by western blots. Results in western blots were quantified using ImageJ and presented as a histogram. H, I Circ_0001810 upregulated AK2 expression in primary neurons. J The plasmid construction of sh-circ_0001810 and its control. K, L The AK2 expression in SHSY-5Y cells after circ_0001810 knockdown. M Dual luciferase assay examined the bind of miR-1197 to the AK2 3′-UTRs in SHSY-5Y cells. The mutant AK2 3′-UTRs reporter and miR-NC mimic were used as negative controls. N, O The AK2 expression in SHSY-5Y cells post-transfection with miR-1197 or the corresponding control. P, Q The AK2 expression in SHSY-5Y cells co-transfected with circ_0001810 and miR-1197. 3′-UTRs, 3′-untranslated regions; Mut, mutant; NC, negative control; WT, wild type. Each bar represented the mean ± SD of three independent experiments. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001