Fig 2: Expression of Robo1 in different segments of the intestines of children with HD (immunohistochemical staining, 400× magnification). (A) Robo1 was abundantly expressed in normal nerve clusters, as indicated by dark brown staining. (B) Robo1 was expressed at a low level in the transitional group, as indicated by light yellow staining. (C) Robo1 was not expressed in the spastic segment group. The localization of the Robo1 protein is shown by the red arrow in the figure. HD = Hirschsprung disease, Robo1 = Roundabout 1.

Fig 3: Slit2 and Robo1 protein expression in the 3 groups (30 tissues per group). (A) Electrophoretograms showing Slit2 and Robo1 protein expression in the 3 groups. The highest levels of the Slit2 and Robo1 proteins were observed in the normal segment group, the lowest expression levels of Slit2 and Robo1 were detected in the spastic segment group, and the expression levels of Slit2 and Robo1 in the transitional segment group were between those in the normal segment group and those in the spastic segment group. (B) Histogram of protein expression in the different groups. The pairwise comparison revealed that the difference was significant; *P < .05. 1 and 2, the normal segment group; 3 and 4, the transitional segment group; 5 and 6, the spastic group. Robo1 = roundabout 1, Slit2 = slit guidance ligand 2.

Fig 4: ROBO1 is a key downstream gene of miR-588. A Sequence of the miR-588 binding site in ROBO1, the miR-588 binding site in the ROBO1 3'-UTR was predicted using Targetscan. B Results of 3'-UTR luciferase assay.. A 3'-UTR vector and miR-588 or miRNA negative control were co-transfected into U87MG cells. The luciferase activity levels were compared with those of the miRNA negative control-transfected cells, which were normalized to 1. C Patients with glioma expressing a high level of ROBO1 experienced significantly shorter survival. D ROBO1 expression was significantly increased in gliomas compared with normal brain tissues. E Cell viability assay of U87MG cells transfected with NC and ROBO1 siRNA. F The ROBO1 protein levels in U87MG cell transfected with miR-588. GAPDH was used as a loading control. G The ROBO1 protein levels in A172 cell transfected with miR-588. H The ROBO1 protein levels in U87MG cells stably transfected with NC, mimics and inhibitor and transfected with ROBO1 siRNAs. GAPDH was used as a loading control

Fig 5: The invasive, migratory and VM-forming abilities of glioma cells were decreased after ROBO1 knockdown. A and E Wound-healing assays of miR-588 inhibitor- and mimics-transfected U87MG cells co-transfected with ROBO1 siRNA. B and F Effects of ROBO1 siRNA on miR-588 inhibitor- and mimics-transfected U87MG cells were examined by Transwell migration assays. C and G Effects of ROBO1 siRNA on miR-588 inhibitor- and mimics-transfected U87MG cells were examined by Transwell invasion assays. D and H Effects of ROBO1 siRNA on miR-588 inhibitor- and mimics-transfected U87MG cells were examined by VM formation assays. **p < 0.01, *p < 0.05 by Kruskal–Wallis one-way ANOVA