Impact of Cullin5-mediated PD-L1 ubiquitination degradation on the radiosensitivity of glioblastoma multiforme
10.3760/cma.j.cn113030-20231008-00119
- VernacularTitle:Cullin5介导PD-L1泛素化降解对胶质母细胞瘤放射敏感性的影响
- Author:
Jianye WANG
1
;
Haidong GAO
;
Rongjun LIU
;
Xinjun WANG
;
Jixin SHOU
Author Information
1. 郑州大学第五附属医院神经外科，郑州 450000
- Keywords:
Glioblastoma;
Ubiquitination degradation;
Radiosensitivity;
Programmed death-ligand 1
- From:
Chinese Journal of Radiation Oncology
2024;33(7):655-660
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the impact of Cullin5 on the radiosensitivity of glioblastoma multiforme cell and its underlying mechanisms.Methods:Glioblastoma U251 cells were transfected with flag-NC plasmids and overexpression plasmids (flag-Cullin5), and divided into the flag-NC group and flag-Cullin5 group. The expression levels of Cullin5 and programmed death ligand 1 (PD-L1) mRNA and protein were assessed using real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. After exposing U251 cells to 0, 2, 4, and 8 Gy X-ray radiation, cell viability was evaluated via the CCK-8 assay, and apoptosis rates were measured using flow cytometry. Co-immunoprecipitation was used to detect the interaction between Cullin5 and PD-L1, and ubiquitination assays were conducted to investigate the ubiquitination effect of Cullin5 on PD-L1. Cells were further transfected with PD-L1 overexpression (oe-PD-L1) and flag-Cullin5 and assigned into the flag-Cullin5 group, flag-Cullin5+oe-NC group, and flag-Cullin5+oe-PD-L1 group. qRT-PCR and Western blot were used to measure the expression levels of Cullin5 and PD-L1 mRNA and protein post-X-ray radiation in these groups. Cell survival rates and apoptosis rates were measured following radiation. Statistical differences between two or among multiple groups were analyzed using LSD- t test or one-way ANOVA. Results:Compared with the flag-NC group, the flag-Cullin5 group showed increased expression levels of Cullin5 mRNA and protein, and decreased PD-L1 protein expression (all P<0.05). Following radiation doses of 4 and 8 Gy, there was a reduction in cell survival rates and an increase in apoptosis rates (both P<0.05). Co-immunoprecipitation assay revealed the presence of PD-L1 in the Cullin5 immunoprecipitate and vice versa. Ubiquitination levels of PD-L1 protein were higher in the flag-Cullin5 group compared to those in the flag-NC group. Additionally, compared to the flag-Cullin5 group, the flag-Cullin5+oe-PD-L1 group exhibited increased PD-L1 mRNA and protein levels (both P<0.05), and following radiation doses of 4 and 8 Gy, these cells showed higher survival rates and lower apoptosis rates (both P<0.05). Conclusion:Cullin5 enhances the radiosensitivity of glioblastoma multiforme cells to radiation therapy through mediating the ubiquitination degradation of PD-L1.
