Fig. 3

MiR-185-5p negatively regulated HOXB5 expression and suppressed the proliferation of GSCs. a Identification of a miRNA that potentially regulated HOXB5 expression based on microRNA, miRDB, Starbase, and TargetScan databases. b, c and d, e qRT-PCR (b, c) and western blotting (d, e) showed HOXB5 expression in GSCs after miR-185-5p mimic or inhibitor treatment. (GSC406: p < 0.0001; GSC201: p < 0.0001; Student’s t-test). f Schematic diagram of the putative miR-185-5p binding site in the 3′-UTR of HOXB5. g The luciferase reporter assays showed that miR-185-5p mimic or inhibitor affected the luciferase activities of HOXB5 in GSCs. (GSC406: p < 0.0001; GSC201: p = 0.0037; Student’s t-test). h The relative expression correlation between miR-185-5p and HOXB5 in 70 cases of glioma patients were detected by qRT-PCR. (Total: r = − 0.4565, p = 0.0430; Grade II: r = − 0.4782, p = 0.0156; Grade III: r = − 0.5146, p = 0.0085; Grade IV: r = − 0.6336, p < 0.0001; Pearson’s correlation analyses). i and j MTS assays showed that miR-185-5p mimic or inhibitor treatment affected the cell viability of GSCs and was reversed by HOXB5 overexpression or knockdown, respectively. (GSC406: p < 0.0001; GSC201: p < 0.0001; One-Way ANOVA). k The EDU assays showed that miR-185-5p mimic or inhibitor treatment affected the proliferation of GSCs and was reversed by HOXB5 overexpression or knockdown, respectively. Scale bar = 100 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). l The neurospheres formation assays showed that miR-185-5p mimic or inhibitor treatment affected the relative size of the neurospheres of GSCs and was reversed by HOXB5 overexpression or knockdown, respectively. Scale bar = 20 μm. (GSC406: p < 0.01; GSC201: p < 0.01; One-Way ANOVA). m and n Limiting dilution assays showed that miR-185-5p mimic or inhibitor treatment affected the neurosphere-forming capacity of GSCs and was reversed by HOXB5 overexpression or knockdown, respectively (GSC406: p < 0.01; GSC201: p < 0.01; ELDA analysis; circles represent corresponding points, triangles mean the point is outside of the log fraction number wells). 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