Fig. 2

EIF4A3-induced circSTX6 promotes metastasis of BCa in vitro and in vivo. A The binding sites for EIF4A3 in the flanking sequences of STX6 pre-mRNA were predicted using CircInteractome database. The primers were used to detect the levels of STX6 pre-mRNA. B RIP assay was performed to identify the putative binding sites of EIF4A3 in the STX6 pre-mRNA. IgG were used as the negative controls. C The relative expression of EIF4A3 in BCa samples and their para-cancer tissues were detected by qRT-PCR. P values are calculated by paired two-sided t-test. D The correlation between the expression of EIF4A3 and circSTX6 in clinical BCa samples. E-F CircSTX6 expression was detected in BCa cells after EIF4A3 down-regulation (E) or up-regulation (F) by qRT-PCR. G The knockdown efficiency of circSTX6 in EJ cells was detected by qRT-PCR. H The overexpression efficiency of circSTX6 in EJ cells was detected by qRT-PCR. I Representative and quantified results of the Transwell migration and invasion assays in EJ cells transfected with scramble, sh-circ#1, sh-circ#2, vector or circSTX6. Scale bar, 100 μm. J-K Cell migration capability of EJ and UMUC3 cells transfected with scramble, shcirc#1, shcirc#2, vector or circSTX6 was evaluated by wound healing assays. Scale bar, 400 μm. L Representative bioluminescence images showed the pulmonary metastasis focuses in the nude mice (n = 5 per group). M H&E staining was performed for histological confirmation of metastasizing tumor in lung. Scale bars, 200 μm. N Kaplan-Meier curves analysis showed the effect of knockdown circSTX6 on the survival of the above nude mice. The data are presented as the means ± S.D. of at least three independent experiments. *P < 0.05. P values are calculated by Student’s t test in B, E, F, H, I, J and K and one-way ANOVA in G, I, J, K and M