Fig. 6

HOXB5 transcriptionally regulated IL6 expression and regulated the proliferation of GSCs via JAK2/STAT3 signaling. a TCGA and CGGA datasets showed that higher HOXB5 expression was associated with enrichment of IL6-mediated JAK2/STAT3 signaling. b The relative expression correlation between HOXB5 and IL6 in 70 cases of glioma patients was detected by qRT-PCR. (Total: r = 0.6160, p < 0.0001; Grade II: r = 0.4548, p = 0.0439; Grade III: r = 0.4723, p = 0.0171; Grade IV: r = 0.5090, p = 0.0094; Pearson’s correlation analyses). c, d, l, m qRT-PCR (c), ELISA (d), and western blotting (l, m) showed the IL6 expression was altered after HOXB5 knockdown or overexpression in GSCs. (c: GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA; d: GSC406: p < 0.001; GSC201: p < 0.01; One-Way ANOVA). e Sequence motif representing the consensus HOXB5 binding motif (JASPAR database), and Schematic diagram of the putative HOXB5 binding site in the 3′-UTR of IL6. f The luciferase reporter assays showed that HOXB5 knockdown or overexpression affected the luciferase activities of IL6 in GSCs. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). g The ChIP qRT-PCR showed that HOXB5 bound to the promoter of IL6. (GSC406: p < 0.01; GSC201: p < 0.001; One-Way ANOVA). h MTS assays showed that HOXB5 knockdown or overexpression affected the cell viability of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. (GSC406: p < 0.001; GSC201: p < 0.001; One-Way ANOVA). i The EDU assays showed that HOXB5 knockdown or overexpression affected the proliferation of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. Scale bar = 100 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). j The neurospheres formation assays showed that HOXB5 knockdown or overexpression affected the relative size of the neurospheres of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. Scale bar = 20 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). k Limiting dilution assays showed that HOXB5 knockdown or overexpression affected the neurosphere-forming capacity of GSCs and was reversed by additional recombinant IL6 or anti-IL6, respectively. (GSC406: p < 0.05; GSC201: p < 0.05; ELDA analysis; circles represent corresponding points, triangles mean the point is outside of the log fraction number wells). l and m Western blotting showed the expression of downstream targets of the IL6-mediated JAK2/STAT3 signaling pathway with HOXB5 knockdown or overexpression in GSCs. EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data were expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001