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Loss of p53 Concurrent with RAS and TERT Activation Induces Glioma Formation

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Abstract

There is an ongoing debate regarding whether gliomas originate due to functional or genetic changes in neural stem cells (NSCs). Genetic engineering has made it possible to use NSCs to establish glioma models with the pathological features of human tumors. Here, we found that RAS, TERT, and p53 mutations or abnormal expression were associated with the occurrence of glioma in the mouse tumor transplantation model. Moreover, EZH2 palmitoylation mediated by ZDHHC5 played a significant role in this malignant transformation. EZH2 palmitoylation activates H3K27me3, which in turn decreases miR-1275, increases glial fibrillary acidic protein (GFAP) expression, and weakens the binding of DNA methyltransferase 3A (DNMT3A) to the OCT4 promoter region. Thus, these findings are significant because RAS, TERT, and p53 oncogenes in human neural stem cells are conducive to a fully malignant and rapid transformation, suggesting that gene changes and specific combinations of susceptible cell types are important factors in determining the occurrence of gliomas.

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Data Availability

All data generated or analyzed during this study are included within this article.

Abbreviations

ABE:

Acyl–biotin exchange assay

CDKN1B:

Cyclin-dependent kinase inhibitor 1 B

ChIP:

Chromatin immunoprecipitation

CNS:

Central nervous system

DNMT:

DNA methyltransferase

FACS:

Fluorescence activating cell sorter

HOXA5:

Homeobox A5

MHC:

Major histocompatibility complex

NF1:

Neurofibrin 1

NSC:

Neural stem cell

PBMCs:

Peripheral blood mononuclear cells

RUNX3:

Runt-related transcription factor 3

RT-PCR:

Real-time polymerase chain reaction

TERT:

Telomerase reverse transcriptase

ZDHHC:

Zinc finger DHHC domain-containing protein

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Acknowledgements

We thank the members of the technical assistance team at the Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences.

Funding

This research was supported by the National Natural Science Foundation of China (grant numbers 82172663, 82104208, 81872066, 81773131, and 81972635), the Innovative Program of Development Foundation of Hefei Centre for Physical Science and Technology (grant number 2021HSC-CIP011).

Author information

Author notes

  1. Meiting Gong and Xiaoqing Fan contributed equally to this work.

Authors and Affiliations

  1. Department of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, No. 81, Meishan Road, Hefei, 230032, Anhui, China

    Meiting Gong, Huihan Yu, Hongzhi Wang & Xueran Chen

  2. Department of Laboratory Medicine, Hefei Cancer Hospital, Chinese Academy of Sciences, No. 350, Shushan Hu Road, Hefei, 230031, Anhui, China

    Meiting Gong, Hongzhi Wang & Xueran Chen

  3. Department of Anesthesiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, No. 17, Lu Jiang Road, Hefei, 230001, Anhui, China

    Xiaoqing Fan

  4. Anhui Province Key Laboratory of Medical Physics and Technology; Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, No. 350, Shushan Hu Road, Hefei, 230031, Anhui, China

    Xiaoqing Fan, Wanxiang Niu, Suling Sun, Hongzhi Wang & Xueran Chen

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  1. Meiting Gong
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Contributions

XQF, HZW, and XRC conceived and designed the experiments; MTG, XQF, WXN, HHY, and SLS performed the experiments; XQF, MTG, and XRC analyzed the data; MTG, XQF, and HHY wrote the paper. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Hongzhi Wang or Xueran Chen.

Ethics declarations

Ethics Approval and Consent to Participate

The study was approved by the Institutional Review Board of the Hefei Cancer Hospital, Chinese Academy of Sciences. All animal experiments were performed in accordance with the guidelines of the Animal Use and Care Committees at the Hefei Institutes of Physical Science, Chinese Academy of Sciences.

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Not applicable.

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The authors declare no competing interests.

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Meiting Gong and Xiaoqing Fan serve as co-first authors.

Supplementary Information

Additional file 1:

Supplementary Figure 1: Gene alterations in glioma using TCGA database. (A) OncoPrint graphs represent p53, EGFR, IDH1, RB1, TERT, and RAS alteration in 871 gliomas using TCGA database. (B) Graphs represent p53 and RAS as well as p53 and TERT co-alteration in 871 gliomas using TCGA database. Supplementary Figure 2: Western blot for p53, EGFR, and RAS in hNSCs transduced with the indicated lentiviral vectors. Supplementary Figure 3: Immunological features of RASG12V/p53-/-/TERT human neural stem cells. (A) Western blot for TNFR2 in wild-type and RASG12V/p53-/-/TERT (Conoly #1, #2 and #3) human neural stem cells (hNSCs). β-tubulin was used as the loading control. (B) Messenger RNA levels of HLA-A and HLA-DRA in wild-type and RASG12V/p53-/-/TERT (Conoly #1, #2 and #3) hNSCs were analyzed by RT-PCR. β-Actin was used as the loading control. (C) Cell viabilityof peripheral blood mononuclear cells (PBMCs) co-cultured with wild-type or RASG12V/p53-/-/TERT hNSCs was determined using the CCK-8 assay. PBMCs were isolated and divided into three groups. In the negative control group, only PBMCs were cultured alone. Wild-type or RASG12V/p53-/-/TERT hNSCs (105/mL) were co-cultured with PBMCs (103/mL). Data represent the mean ± SD of three separate experiments. (D) Expression level of MHC, HLA-ABC, and HLA-DR in wild-type or RASG12V/p53-/-/TERT hNSCs was determined by FACS. Supplementary Figure 4: The summary data for Fig. 3C. Data from three independent experiments were shown. Supplementary Figure 5: The summary data for Fig. 3F. Data from three independent experiments were shown. Supplementary Figure 6: The summary data for Fig. 4A. Data from three independent experiments were shown. Supplementary Figure 7: The summary data for Fig. 4E. Data from three independent experiments were shown. Supplementary Figure 8: Negative contribution of miR-1275 to GFAP expression by targeting the 3’ -UTR. (A) Left panel: the predicted binding site for miR-1275 in the GFAP 3'-UTR is indicated (arrow). The complementary sequence between GFAP and miR-1275 is indicated above the arrow. The nucleotide position of the targsite is indicated relative to the position of the stop codon of GFAP (the first nucleotide after the stop codon of GFAP is defined as 1). Right panel: Scheme of reporter vectors; wild-type GFAP 3'-UTR sequence (upper) and mutant GFAP 3'-UTR sequence (lower) are shown. Black triangles indicate the predicted miR-1275 binding site within the 3'-UTR of the GFAP gene. (B) Luciferase reporter assay with GFAP-3'-UTR reporter vector and miRNA fragment at hNSC. Luciferase values are relative to Renilla luciferase activity (n = 3). Error bars indicate SD. statistical analysis was performed with one-way ANOVA. 

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Gong, M., Fan, X., Yu, H. et al. Loss of p53 Concurrent with RAS and TERT Activation Induces Glioma Formation. Mol Neurobiol 60, 3452–3463 (2023). https://doi.org/10.1007/s12035-023-03288-w

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