Fig. 4

circVAPA functions as a sponge for miR-377-3p and miR-494-3p. a Anti-Ago2 RNA IP assay for detecting circVAPA in DMS273 cells. Anti-IgG was used as a negative control. b Anti-Ago2 RIP was performed in DMS273 cells. CircHIPK3 was a positive control, while EIciPAIP2 was a negative control. c Predicted putative binding miRNAs of circVAPA with CircInteractome. d Illustration of the experimental procedure for the circVAPA pull-down assay with biotinylated antisense oligonucleotides. e RNA pull-down efficiency of circVAPA in DMS273 cells. The enrichment of the circVAPA probe or negative probe was detected by RT-qPCR assay to analyze potential miRNAs associated with circVAPA. Negative probe, a biotin-labeled oligonucleotide with scrambled sequences; circVAPA probe, a biotin-labeled oligonucleotide with antisense sequences targeting the circVAPA junction site. f Anti-Ago2 RIP was performed in DMS273 cells transiently overexpressing mimics NC/miR-377-3p/miR-494-3p to detect RNA enrichment in IP complexes. Anti-IgG was used as a negative control. The expression of circVAPA and miR-377-3p/miR-494-3p were detected by RT-qPCR. g The relative luciferase activities were detected in 293T cells after co-transfection with Lu-circVAPA-WT or Lu-circVAPA-Mut and NC or miR-377-3p/miR-494-3p mimics, respectively (left). Lu-circVAPA-DM represents Lu-circVAPA-Mut 1 & 2, which contains two mutation sites. The firefly luciferase activities were measured and normalized to renilla luciferase activities (F/R). Wild-type (WT) and mutated-type (MuT) sequences of the putative binding sites between circVAPA and miR-377-3p/miR-494-3p were listed (right). h–k Colony formation assay of DMS273 cells and H82 cells transiently transfected with siRNAs, plasmids, miRNA inhibitors or mimics as indicated. miR-377 inhi, miR-377 inhibitor; miR-494 inhi, miR-494 inhibitor. SCR, siRNA with scrambled sequences; si-circVAPA, the co-transfection of two independent siRNAs target circVAPA; EV, the empty vector; circVAPA, the circVAPA overexpression plasmid; miR-377 mimic/miR-494 mimic, transiently overexpressing miR-377-3p/miR-494-3p, respectively; miR-377 inhibitor/miR-494 inhibitor, transiently suppressing miR-377-3p/miR-494-3p, respectively. (All data are presented as the mean ± SD; ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student’s t-test). Three independent assays were performed in the above assays. *** miR-377/miR-494 in this article represents miR-377-3p/miR-494-3p, respectively