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. 2020 Apr 28;55:102736. doi: 10.1016/j.ebiom.2020.102736

Fig. 4.

Fig. 4

miR-603 suppresses glioblastoma stem-cell state through down-regulation of IGF1. (a) miR-603 suppressed expression of the stem cell marker, Sox2. Representative immunofluorescence staining images of Sox2 (green) in the patient-derived glioblastoma neurosphere line CMK3 transfected with miR-603 or miR-NT. Studies were performed in the presence or absence of IGF1 treatment. DAPI (blue) staining was used to visualize nuclei. Quantifications of Sox2-positive cells were provided. ***p < 0.001 between indicated groups (one-way ANOVA). Scale bar represents 100 μm. (b) and (d) The mRNA levels of stem cell markers Sox2 (b) and Olig2 (d) were suppressed by miR-603 transfection in CMK3 cells, which was reversed by exogenous addition of IGF1. *p < 0.05 and **p < 0.01 between indicated groups (one-way ANOVA). (c) Olig2 expression in CMK3 cells transfected with miR-NT or miR-603 was assessed using western blotting. (e) miR-603 transfection induced expression of the differentiation marker, GFAP. Representative immunofluorescence staining images of GFAP (green) in CMK3 cells transfected with miR-603 or miR-NT in the presence or absence of IGF1 treatment. Quantifications of GFAP-positive cells were provided. ***p < 0.001 between indicated groups (one-way ANOVA). Scale bar represents 100 μm. (f) and (g) In vitro limiting dilution assay was performed in CMK3 cells transfected with miR-603 or miR-NT in the presence or absence of IGF1 treatment. Frequencies of clonogenic cells in each group were determined by ELDA analysis (g). **p < 0.01 between indicated groups (one-way ANOVA). Representative images of neurospheres formed in indicated experimental conditions were shown (f). Scale bar: 10 µm. (h) miR-603 transfection suppressed tumorigenicity of patient-derived glioblastoma neurosphere line BT-83. BT-83(vector) or BT-83(IGF1) cells transfected with miR-NT or miR-603 were injected into the subcutaneous compartment of nude mice (10,000 cells per injection). Twenty injections were performed for each experimental condition. *p < 0.05, ***p < 0.001 between indicated groups (Chi-square test).