Figures & data
Table 1. Clinicopathological feature of the enrolled patients
Figure 1. RUNX1 is highly expressed in GBM and associated with a poor prognosis (a) The expression of RUNX1 in GBM tumors and normal brain tissues was compared using data from GEPIA database. (b-d) Relative mRNA and protein level of RUNX1 in primary and recurrent GBM samples, analyzed by qRT-PCR (b), Western blot (c) and immunochemistry (d) E. Kaplan–Meier survival assessment of GBM patients, which were divided into low and high RUNX1 expression groups (n = 76 in each group, P < 0.05). Each sample was analyzed in 3 independent experiments. * P < 0.05 and *** P < 0.001
Figure 2. RUNX1 is required for TMZ resistance in GBM cells (a-b) mRNA and protein levels of RUNX1 in U87 and A172 GBM cell lines as compared to astrocytes C8-DA. (c) Western blot analysis of RUNX1 in TMZ-sensitive parental cells and TMZ-resistant cells. (d) The level of RUNX1 in U87 and A172 cells was analyzed after being transfected with RUNX1-siRNA and control siRNA (si-NC). (e-g) Cell viability (e-f) and IC50 (g) of RUNX1-silenced cells was compared to the cells treated with si-NC. Cells were treated with different TMZ concentration (0, 1 μM, 5μM, 25 μM, 50 μM, 100 μM, 24 h), and trypan blue assay was performed to quantify the viable cells. Each sample was repeated in at least 3 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001
Figure 3. RUNX1 regulates tumor cell proliferation, migration and invasion. (a-b) The knockdown or overexpression of RUNX1 after the transfection with si-RUNX1 or pcDNA-RUNX1 expression vector was analyzed by Western blot and qRT-PCR in U87 and A172 cells. (c) CCK-8 assay in cells after transfection with si-NC, si-RUNX1, Empty vector or pcDNA-RUNX1. (d) Transwell migration assay, (e) Transwell invasion assay, (f) colony formation assay, (g) apoptosis were performed in U87 and A172 cells after transfection with si-NC, si-RUNX1, Empty vector or pcDNA-RUNX1. Each sample was repeated at least 3 times. * P < 0.05, ** P < 0.01, and *** P < 0.001
Figure 4. RUNX1 is a direct target of miR-128-3p. (a) Alignment of the miR-28-3p sequence with that of RUNX1 3ʹ-UTR. (b) Dual luciferase reporter assay was performed in U87 and A172 cells in the presence of miR-NC or miR-128-3p. WT: wildtype reporter containing binding site. MUT: reporter with mutated binding site. (c) The expression of miR-28-3p in TMZ-sensitive parental cells and TMZ-resistant cells. (d) Expression level of miR-28-3p in TMZ-resistant cells after transfection with miR-128-3p mimic or miR-128-3p inhibitor. (e) Gene and protein expression levels of RUNX1 in TMZ-resistant cells transfected miR-128-3p mimic or miR-128-3p inhibitor. (f) Fluorescence in situ hybridization (FISH) was performed using miR-128-3p probe + control probe or miR-128-3p probe + RUNX1 probe to investigate the co-localization of miR-128-3p and RUNX1 in GBM cell lines. Each sample was repeated at least 3 times. *** P < 0.001
Figure 5. MiR-128-3p attenuates TMZ resistance in GBM by targeting RUNX1 (a-b) Cell viability and IC50 of cells transfected with miR-128-3p mimic or miR-NC in the increasing concentration of TMZ treatment (0, 1 μM, 5 μM, 25 μM, 50 μM, 100 μM, 24 h). (c) qRT-PCR analysis of RUNX1 expression in U87 and A172 cells transfected with miR-NC, miR-128-3p mimic and miR-128-3p mimic + pcDNA-RUNX1. (d) Cell viability of U87 and A172 cells transfected with miR-NC, miR-128-3p mimic and miR-128-3p mimic + pcDNA-RUNX1 after TMZ treatment (50 μM) for 0 h, 24 h, 48 h and 72 h. (e) Colony formation assay, (f) Transwell migration assay, (g) Transwell invasion assay were performed in U87 and A172 cells transfected with miR-NC, miR-128-3p mimic and miR-128-3p mimic + pcDNA-RUNX1 after TMZ treatment (0, 50 μM, 100 μM). Each experiment was repeated at least 3 times. * P < 0.05, ** P < 0.01, and *** P < 0.001
Figure 6. RUNX1/miR-128-3p axis regulates MRP1 expression in TMZ-resistant GBM cells. (a-b) Gene and protein level of MRP1 in TMZ-sensitive parental cells and TMZ-resistant cells were detected by qRT-PCR and Western blot. (c-d) Gene and protein levels of MRP1 were examined in cells transfected with miR-NC, miR-128-3p mimic, miR-128-3p mimic+ pcDNA-RUNX1 or si-NC, si-RUNX1 and si-RUNX1+ miR-128-3p inhibitor. Each sample was repeated at least 3 times. *** P < 0.001
