Fig 1.
SFRP2 is downregulated in radiotherapy treated glioma patients and predicted poor prognosis.
A, the differentially expressed mRNAs in radiotherapy-treated glioma patients versus non-radiotherapy treated glioma patients in TCGA were depicted in the volcano plots. B, SFRP2 expression in radiotherapy-treated glioma patients and non-radiotherapy treated glioma patients in TCGA. C, SFRP2 expression in WHO grade II, III and IV glioma patients in TCGA. D, overall survival of glioma patients according to SFRP2 expression in TCGA. E, progression-free survival of glioma patients according to SFRP2 expression in TCGA. F, SFRP2 expression in radiotherapy-treated glioma patients and non-radiotherapy treated glioma patients in the validated cohort of our study. G, SFRP2 expression in WHO grade II, III and IV glioma patients in the validated cohort of our study. H, overall survival of glioma patients according to SFRP2 expression in the validated cohort of our study. I, progression-free survival of glioma patients according to SFRP2 expression in the validated cohort of our study. J, IHC staining of SFRP2 in radiotherapy-treated glioma tissue and non-radiotherapy treated glioma tissue. K, SFRP2 expression in glioma cell lines or normal glia tissue was evaluated by qRT-PCR. **P< 0.001.
Table 1.
Correlation between SFRP2 expression and clinicopathological characteristics of glioma patients.
Fig 2.
SFRP2 knockdown promotes soft agar colony formation, cancer stemness and radioresistance of glioma cells.
A, SW1783 and SW1088 cells were introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles, then protein expression of SFRP2 was evaluated by western blot. B-C, SW1783 and SW1088 cells were introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles, then seeded in 6-well plates at 8000/well for soft agar assay. Representative plates (B) and average number of colonies per well (C) were shown. D-E, SW1783 and SW1088 cells were introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles, then seeded in 6-well plates at 3000/well for sphere formation assay. Representative images (D) and average number of spheres per image (E) were shown. F, expression of Oct4, Lin28, Nanog and Sox2 in SW1783 and SW1088 cells introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles was evaluated by qRT-PCR. G, SW1783 and SW1088 cells were introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles, then seeded in 96-well plates (3000/well) and treated with a single dose of 2 or 4 Gy X-ray irradiation. Cell viability was tested at 72 h after the irradiation. H-J, SW1783 and SW1088 cells infected with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles were seeded in 6-well plates (1 × 106/well) and treated with a single dose of 2 or 4 Gy X-ray irradiation. Then cells were used for TUNEL staining (H-I) or western blot (J) at 24 h after irradiation. Representative images (H) and percentages of TUNEL positive cells (I) were shown. K-M, SW1783 cells (2 × 106) infected with sg-SFRP2-1 or sg-NC lentiviral particles were subcutaneously injected into nude mice. Tumor xenografts were treated with/without 2 Gy X-ray irradiation daily for three weeks from day 7. Tumor growth curves (K), image of tumor xenografts (L) and tumor weight (M) were shown. *P< 0.05, **P< 0.001.
Fig 3.
Forced SFRP2 expression suppresses soft agar colony formation, cancer stemness and radioresistance of glioma cells.
A, LN18 and T98G cells were infected with SFRP2 expression lentivirus or empty vector (EV) control, then protein expression of SFRP2 was evaluated by western blot. B-C, LN18 and T98G cells were infected with SFRP2 expression lentivirus or EV control, then seeded in 6-well plates at 8000/well for soft agar assay. Representative plates (B) and average number of colonies per well (C) were shown. D-E, LN18 and T98G cells were infected with SFRP2 expression lentivirus or EV control, then seeded in 6-well plates at 3000/well for sphere formation assay. Representative images (D) and average number of spheres per image (E) were shown. F, expression of Oct4, Lin28, Nanog and Sox2 in LN18 and T98G cells infected with SFRP2 expression lentivirus or EV control was evaluated by qRT-PCR. G, LN18 and T98G cells were infected with SFRP2 expression lentivirus or EV control, then seeded in 96-well plates (3000/well) and treated with a single dose of 2 or 4 Gy X-ray irradiation. Cell viability was tested at 72 h after irradiation. H-J, LN18 and T98G cells infected with SFRP2 expression lentivirus or EV control were seeded in 6-well plates (1 × 106/well) and treated with a single dose of 2 or 4 Gy X-ray irradiation. Then cells were used for flow cytometry (H-I) or western blot (J) at 24 h after the irradiation. Percentages of Annexin-V positive cells (I) were shown. *P< 0.05, **P< 0.001.
Fig 4.
Wnt signaling is activated in radiotherapy treated glioma patients.
A, the expression of indicated genes in TCGA database. B, the expression of indicated genes in the Validation cohort of our study. C-D, the expression of active β-catenin in radiotherapy treated and non-radiotherapy treated glioma samples was evaluated by IHC staining (C) or western blot (D). *P< 0.05, **P< 0.001.
Fig 5.
SFRP2 regulates Wnt/β-catenin activation in glioma cells.
A-B, SW1783 and SW1088 cells were introduced with sg-SFRP2-1, sg-SFRP2-2 or sg-NC lentiviral particles, then protein expression of indicated genes was evaluated by western blot (A). Relative protein expression compared with GAPDH (B) was shown. C-D, LN18 and T98G cells were infected with SFRP2 expression lentivirus or EV control, then protein expression of indicated genes was evaluated by western blot (C). Relative protein expression compared with GAPDH (D) was shown. **P< 0.001.
Fig 6.
Pharmacological inhibition of Wnt/β-catenin pathway represses sphere formation, stemness and radioresistance of glioma cells.
A, SW1783 and SW1088 cells infected with sg-SFRP2-1 or sg-NC lentiviral particles were seeded in 6-well plates (1 × 106/well), and treated with 1 μM XAV-939 or equal volume of DMSO (Veh) for 24 h. The protein expression of indicated genes was evaluated by western blot. B-C, SW1783 and SW1088 cells were introduced with sg-SFRP2-1 or sg-NC lentiviral particles, then seeded in 6-well plates at 8000/well for soft agar assay. Cells were treated with 1 μM XAV-939 or equal volume of DMSO (Veh) at the same time. Representative plates (B) and average number of colonies per well (C) were shown. D-E, SW1783 and SW1088 cells were introduced with sg-SFRP2-1 or sg-NC lentiviral particles, then seeded in 6-well plates at 3000/well for sphere formation assay. Cells were treated with 1 μM XAV-939 or equal volume of DMSO (Veh) at the same time. Representative images (D) and average number of spheres per image (E) were shown. F, SW1783 and SW1088 cells introduced with sg-SFRP2-1 or sg-NC lentiviral particles were treated with 1 μM XAV-939 or equal volume of DMSO (Veh) for 6 d, then the expression of Oct4, Lin28, Nanog and Sox2 was evaluated by qRT-PCR. G, SW1783 and SW1088 cells introduced with sg-SFRP2-1 or sg-NC lentiviral particles were seeded in 96-well plates (3000/well) and treated with 2 or 4 Gy X-ray irradiation, 1 μM XAV-939 or equal volume of DMSO (Veh) as indicated. Cell viability was tested at 72 h after the irradiation. H-J, SW1783 cells (2 × 106) infected with sg-SFRP2-1 or sg-NC lentiviral particles were subcutaneously injected into nude mice. Tumor xenografts were treated with 2 Gy X-ray irradiation, 25 mg/kg XAV-939 or equal volume of 50% PEG-400 as indicated daily for three weeks from day 7. Tumor growth curves (H), image of tumor xenografts (I) and tumor weight (J) were shown. *P< 0.05, **P< 0.001. n.s., not significant.