Fig. 3.
miR-603-induced IGF1 suppression enhances glioblastoma sensitivity to ionizing radiation in vitro and in vivo. (a) Stable miR-603 expressing LN340 glioblastoma line exhibited increased sensitivity to IR. Clonogenic survival for two independent clones of LN340 with or without stable ectopic miR-603 expression (labeled as LN340(miR-603) or LN340(miR-Empty), respectively) were determined post IR treatment. *p < 0.05 and **p < 0.01 between LN340(miR-603)−1 and LN340(miR-Empty)−1 or −2 at indicated IR dose. #p < 0.05, ##p < 0.01, ###p < 0.001 between LN340(miR-603)−2 and LN340(miR-Empty)−1 or −2 at indicated IR dose (Student's t-test). (b) Exogenous IGF1 addition suppressed the radiation sensitizing effect of miR-603. Cell viability was determined 5 days after IR and normalized to that of LN340(miR-Empty)−1 cells without IR or IGF1 treatment. *p < 0.05 and **p < 0.01 between indicated groups (Student's t-test). (c) miR-603 expression increased comet tail moment in response to IR. Comet assay was performed in LN340(miR-Empty)−1 and LN340(miR-603)−1 cells (unirradiated or received 6 Gy IR) with or without IGF1 treatment. Representative images of olive moment and the quantification were shown. Results were normalized to comet tail moment in unirradiated LN340(miR-Empty)−1 cells without IGF1 treatment. ***p < 0.001 between indicated groups (Student's t-test). Scale bar is 50 μm. (d) miR-603 expression enhanced γ-H2AX foci accumulation in response to IR. Representative immunofluorescence images of γ-H2AX foci in LN340(miR-Empty)−1 and LN340(miR-603)−1 cells (unirradiated or received 6 Gy IR) with or without IGF1 treatment were shown (left panel). Quantification of γ-H2AX foci was provided (right panel). Results were normalized to γ-H2AX foci in unirradiated LN340(miR-Empty)−1 cells without IGF1 treatment. *p < 0.05 and ***p < 0.001 between indicated groups (Student's t-test). Scale bar is 5 μm. (e) and (f) Radiation sensitizing effects of miR-603 were epistatic to that of IGF1R silencing. The effects of radiation alone or combination with IGF1R silencing on colony formation in LN340(miR-Empty)−1 or −2 (e) and LN340(miR-603)−1 or −2 cells (f) were shown. * p < 0.05 between LN340(miR-Empty)−1+siIGF1R and LN340(miR-Empty)−1 or −2+control siRNA at indicated IR dose. # p < 0.05 between LN340(miR-Empty)−2+siIGF1R and LN340(miR-Empty)−1 or −2+control siRNA at indicated IR dose (Student's t-test). (g) and (h) Ectopic IGF1 expression suppressed radiation sensitizing effect of miR-603 in vivo. Subcutaneous xenograft tumor growth curves were plotted after tumor cell inoculation. BT-83(vector) or BT-83(IGF1) xenografts were injected with human miR-603 mimic or miR-NT twice at 24-hour interval after the tumor volume reached 50mm3. IR (3 Gy/day) was administered for 2 consecutive days after the last dose of miRNA injection. Panel g and panel h are derived from the same experiment but plotted separately for better visualization of the data. *p < 0.05, ***p < 0.001 between indicated groups (one-way ANOVA). (i) Kaplan–Meier survival curves of mice bearing various intracranial BT-83 implants with or without radiation treatment (2 Gy/day for 5 consecutive days). Implanted glioblastoma neursophere lines included: BT-83(vector), BT-83(IGF1)(with stable IGF1 expression), BT-83(miR-603)(with stable miR-603 expression), and BT-83(IGF1+miR-603)(with stable IGF1 and miR-603 expression). (j) miR-603 level in clinical glioblastoma specimens correlated with radiation response. Scatter plot of miR-603 expression levels in clinical glioblastoma specimen obtained from glioblastoma patients who underwent repeat radiation therapy due to tumor recurrence. Disease stability or progression were assessed approximately one year post re-radiation. 12 patients achieved stable disease, and 14 patients suffered disease progression. p = 0.0126 compared to progression group (Student's t-test). (k) miR-603 sensitized patient-derived glioblastoma cells to IR. Clonogenic potential of CMK3 cells (propagated as neurospheres) was determined using limiting dilution assays. CMK3 cells were transfected with miR-603 or miR-NT before IR treatment. ***p < 0.001 between indicated groups (Chi-square test).