Knockdown of ATRX enhances radiosensitivity in glioblastoma

Abstract

Background: Glioblastoma are highly malignant type of primary brain tumors. Treatment for glioblastoma multiforme (GBM) generally involves surgery combined with chemotherapy and radiotherapy. However, the development of tumoral chemo- and radioresistance induces complexities in clinical practice. Multiple signaling pathways are known to be involved in radiation-induced cell survival. However, the role of alpha-thalassemia X-linked mutant retardation syndrome (ATRX), a chromatin remodeling protein, in GBM radioresistance remains unclear.

Methods: In the present study, the ATRX mutation rate in patients with glioma was obtained from The Cancer Genome Atlas, while its expression analyzed using bioinformatics. Datasets were also obtained from the Gene Expression Omnibus, and ATRX expression levels following irradiation of GBM were determined. The effects of ATRX on radiosensitivity were investigated using a knockdown assays.

Results: The present study demonstrated that the ATRX mutation rate in patients with GBM was significantly lower than that in patients with low-grade glioma, and that patients harboring an ATRX mutation exhibited a prolonged survival, compared with to those harboring the wild-type gene. Single-cell RNA sequencing demonstrated that ATRX counts increased 2 days after irradiation, with ATRX expression levels also increasing in U-251MG radioresistant cells. Moreover, the results of in vitro irradiation assays revealed that ATRX expression was increased in U-251MG cells, while ATRX knockdown was associated with increased levels of radiosensitivity.

Conclusions: High ATRX expression levels in primary GBM may contribute to high levels of radioresistance. Thus ATRX is a potential target for overcoming the radioresistance in GBM.

Keywords: ATRX; Glioblastoma; Knockdown; Radiosensitivity.

Fig. 1

ATRX mutation in A GBM and B LGG. ATRX mutation rate in patients with GBM (8%) was significantly lower than in patients with LGG (77%). Missense mutations, frameshift deletions, and in-frame insertions were the major mutations observed in ATRX in patients with GBM. ATRX mutations observed in patients with LGG were all missense mutations. ATRX, alpha-thalassemia X-linked mutant retardation syndrome; GBM, glioblastoma multiforme; LGG, low-grade glioma

Fig. 2

Kaplan-Meier analysis and log-rank test of overall survival between ATRX mutation and wild-type group. A Patients with an ATRX mutation exhibited a significantly prolonged survival time compared with patients with the ATRX wild-type gene (P = 0.003). B Patients with an ATRX deletion exhibited a significantly prolonged survival time compared with patients with the ATRX wild-type gene (P = 0.007). No significant differences were observed in patients with ATRX SNPs compared with patients with the ATRX wild-type gene (P = 0.113). ATRX, alpha-thalassemia X-linked mutant retardation syndrome; SNP, single-nucleotide polymorphism

Fig. 3

ATRX expression levels analyzed in GSE162931 and GSE206917 datasets using bioinformatics. A Single-cell RNA sequencing profiling in GSE162931 datasets revealed that ATRX expression levels were increased 2 days after radiation. B Bulk RNA sequencing analysis revealed that ATRX expression levels were increased in recurrent GBM tissue samples; however, the difference was not statistically significant. ****P < 0.0001. ATRX, alpha-thalassemia X-linked mutant retardation syndrome; GBM, glioblastoma multiforme

Fig. 4

ATRX expression levels measured by reverse transcription-quantitative PCR after irradiation and siRNA transfection assays. A and B ATRX expression levels were significantly increased following irradiation in U-251MG cells, and the expression levels peaked at 24 h after radiation. Relative ATRX/GAPDH mRNA expression levels were defined as the ratio of normalized ATRX mRNA levels in the experimental group to the control group. C and D Transfection efficiency of siRNA 1–3 reached 90–60% in U-251MG cells. Transfection efficiency of siRNA 1–3 reached 28–59% in LN229 cells. E and F ATRX expression levels were not increased in all four siRNA groups in U-251MG and LN229 cells following irradiation. *P < 0.05, **P < 0.01, ***P < 0.001. siRNA, small interfering RNA; ATRX, alpha-thalassemia X-linked mutant retardation syndrome

Fig. 5

ATRX knockdown increases the radiosensitivity of U-251MG cells. Colony formation assays were carried out using U-251MG and LN229 cells. Transfection with siRNA 1–3 significantly inhibited U-251MG cell proliferation compared with siRNA-NC. But cell proliferation of LN229 was not inhibited. **P < 0.01. ATRX, alpha-thalassemia X-linked mutant retardation syndrome; siRNA, small interfering RNA; NC, negative control