Construction Of A Glioma Model Based On Gene Mutations In Neural Stem Cells And The Mechanism Of Gliomagenesis

Glioma is a primary intracranial tumor with unique biological and clinical characteristics,and most patients have a poor prognosis after surgery.The cellular origin of glioma has not been thoroughly studied,and understanding the oncogenesis of glioma can not only reveal the oncogenic mechanism of CNS tumors,but also provide new targets and protocols for clinical treatment.Currently,the most recognized theory of tumorigenesis is the intracellular accumulation of proto-oncogene activation and oncogene inactivation.The development of glioma involves various genes,such as TP53,KRAS,IDH 1 and 2,EGFR and TERT,and the establishment of a more natural and appropriate glioma animal model is essential to study the development of glioma more closely to the real glioma process.This study attempted to establish a specific glioma primary model based on the gene mutation characteristics of gliomas.It was found that mutations or aberrant expression of KRAS,TERT and TP53 are associated with glioma development,and these mutated genes can induce human neural stem cells（h NSCs）to develop malignant gliomas.TP53 deletion,KRASG12 V and TERT activation enhance the self-renewal capacity as well as the differentiation potential of h NSCs.KRAS^G12V/TP53^-/-/TERT h NSCs have increased cell viability at the differentiation stage and also have more significant migratory and invasive properties.Genomic enrichment analysis（GSEA）also showed that h NSCs carrying KRAS^G12V/TP53^-/-/TERT had a more pronounced molecular signature of glioma than control h NSCs.ZDHHC family proteins play a key role in gliomagenesis.In KRAS^G12V/TP53^-/-/TERT h NSCs,EZH2 palmitoylation modifications were found to be significantly upregulated.This was attributed to the upregulation of ZDHHC5 expression in mutant h NSCs that increased the level of palmitoylation modification of EZH2 and altered the phosphorylation status of EZH2 S21.Meanwhile,EZH2 suppressed glioma-associated tumor suppressor genes such as CDKN1 B,RUNX3 and HOXA5 by targeting H3K27me3.furthermore,injection of the EZH2 inhibitor Tazemetostat into mice carrying KRAS^G12V/TP53^-/-/TERT h NSCs in mice,tumor growth was found to be significantly inhibited.Glial fibrillary acidic protein（GFAP）was highly co-expressed with H3K27me3 in KRAS^G12V/TP53^-/-/TERT h NSCs.Further studies revealed that mi R-1275 directly negatively regulated GFAP protein expression.Bisulfite genome sequencing（BSP）revealed that the upregulation of OCT4 expression in the self-renewal phase of KRAS^G12V/TP53^-/-/TERT h NSCs was due to the downregulation of the methylation level of the OCT4 promoter.Meanwhile,DNMT3 A,a member of the DNMT family,was found to suppress OCT4 expression,and upregulation of DNMT3 A expression significantly inhibited tumor growth in KRAS^G12V/TP53^-/-/TERT h NSCs.At the stage of gliomagenesis and proliferation,palmitoylation-modified EZH2 increased GFAP expression by activating H3K27me3,which in turn decreased mi R-1275;meanwhile,palmitoylation-modified EZH2 also weakened the binding of DNMT3 A to the OCT4 promoter region and enhanced the transcriptional expression of OCT4.In summary,this study successfully constructed a gliomagenesis model of KRAS^G12V/TP53^-/-/TERT h NSCs and demonstrated that mutations or abnormal expression of KRAS,TERT and TP53 are associated with glioma development.KRAS^G12V/TP53^-/-/TERT h NSCs undergo malignant transformation mainly by ZDHHC5-mediated palmitoylation modification of EZH2 regulating upregulation of GFAP and OCT4 expression,which ultimately leads to malignant cell self-renewal as well as increased differentiation.Therefore,these results provide a new basis for the hypothesis of glioma of neural stem cell origin and some innovative therapeutic ideas for the clinical treatment of glioma.