Abstract
Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, which shows great clinical and biomolecule heterogeneity. Currently, surgery is still the main method of neuroblastoma treatment and specific therapeutic drugs are lacking, so useful targets are urgently needed. TRIM21 is a RING-type E3 ligase that its overexpression promotes the progression of human glioma, while whose effects on neuroblastoma have not been illustrated. Firstly, the shRNAs targeting TRIM21 were designed and found that the ablation of TRIM21 inhibits the proliferation of human neuroblastoma cells. Then the molecular mechanism study indicated that TRIM21 interacts with, and mediates p21 degradation by ubiquitination modification. Further study demonstrates that TRIM21 regulates the proliferation of neuroblastoma cells in a p21-dependent manner. These results suggest that TRIM21 might be a potential therapeutic target for neuroblastoma.
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Funding
This work was funded by the National Natural Science Foundation of China (to Z.R.W, Grant No. 81801367); Medical Health Science and Technology Research Project of Zhejiang Province of China (to Z.R.W, Grant No. 2020RC079); Medical Health Science and Technology Research Project of Zhejiang Province of China (to C.D.W, 2018KY515); Science and Technology Project of Wenzhou City of China (to J.L.L, Y2020061).
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JL designed and supervised the whole project. FW, ZW, QL and ZN performed most of the experiments. All the authors read and approved the manuscript.
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Supplementary file1 (PDF 899 kb) Supplementary Figure 1. The knockout of TRIM21 inhibits the proliferation of SHSY5Y cells. (A) Exploring the role of TRIM21 in brain tumor, TCGA data was mined using the GEPIA (http://gepia.cancer-pku.cn/). GBM, Glioblastoma multiforme; LGG, Brain Lower Grade Glioma; T, Tumor tissue; N, normal tissue. (B and C) A TRIM21-/- SHSY5Y cell line was generated using the CRISPR-CAS9 technique. SHSY5Y cells were transfected with CRISPR-CAS9-based sgRNA, monoclonal were picked and detected by immunoblotting analysis (B), and then genetic ablation of TRIM21 with a 5-bp deletion was confirmed by Sanger sequencing. (D) TRIM21 knockout inhibits the proliferation of SHSY5Y cells. Wild type (WT) and TRIM21-/- SHSY5Y cells were seeded into 96-well plates, and the cells viability was detected using the CCK8 assay at the indicated time points. The data were expressed as mean ± SD and analyzed using one-way ANOVA with Tukey’s post-hoc test. *P < 0.01, **P < 0.01, very significantly different, three independent experiments. (E) TRIM21 knockout inhibits the colony formation of SHSY5Y cells. Wild type (WT) and TRIM21-/- SHSY5Y cells were seeded into 6-well plates. Colonies were fixed with 4% paraformaldehyde and were stained with 0.1% crystal violet 7 days later. The images were acquired using a camera, and the colony numbers were counted and calculated. The data were expressed as mean ± SD and analyzed using student's t-test. **P < 0.01, very significantly different, three samples for each group.
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Wang, F., Wu, Z., Li, Q. et al. Ubiquitination of p21 by E3 Ligase TRIM21 Promotes the Proliferation of Human Neuroblastoma Cells. Neuromol Med 23, 549–560 (2021). https://doi.org/10.1007/s12017-021-08661-3
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DOI: https://doi.org/10.1007/s12017-021-08661-3