Fig. 6.

circVAPA promotes the progression of SCLC through the miR-377-3p and miR-494-3p/IGF1R/AKT axis. a and b Western blot analysis of the effects of circVAPA or miR-377-3p/miR-494-3p on IGF1R, AKT, and its downstream protein expression in SCLC cells. 377 inhi, miR-377 inhibitor, 494 inhi, miR-494 inhibitor; 377 mimic, miR-377 mimic; 494 mimic, miR-494 mimic. c Western blot analysis of the effect of overexpressing circVAPA or silencing IGF1R on AKT and its downstream protein expression in SCLC cells. d Western blot analysis of the effect of overexpressing circVAPA or IGF1R inhibitor (drug BMS-536924) on AKT and its downstream protein expression in SCLC cells. e The relative luciferase activities were detected in 293T cells after co-transfection with Lu-IGF1R-WT and mimic-NC or the circVAPA overexpression plasmid or miR-377-3p/miR-494-3p mimics, respectively. The firefly luciferase activities were measured and normalized to renilla luciferase activities (F/R). f-g Representative images of immunohistochemistry analysis of IGF1R (f) and p-AKT (g) in three independent SCLC cases. Scale bar, 50 μm. 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; si-IGF1R, an independent siRNA targeting IGF1R; IGF1Ri, the addition of IGF1R inhibitor (drug BMS-536924). (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