The promoting effect and mechanism of MAD2L2 on stemness maintenance and malignant progression in glioma

Abstract

Background Glioblastoma, the most common primary malignant tumor of the brain, is associated with poor prognosis. Glioblastoma cells exhibit high proliferative and invasive properties, and glioblastoma stem cells (GSCs) have been shown to play a crucial role in the malignant behavior of glioblastoma cells. This study aims to investigate the molecular mechanisms involved in GSCs maintenance and malignant progression. Methods Bioinformatics analysis was performed based on data from public databases to explore the expression profile of Mitotic arrest deficient 2 like 2 (MAD2L2) and its potential function in glioma. The impact of MAD2L2 on glioblastoma cell behaviors was assessed through cell viability assays (CCK8), colony formation assays, 5-Ethynyl-2ʹ-deoxyuridine (EDU) incorporation assays, scratch assays, and transwell migration/invasion assays. The findings from in vitro experiments were further validated in vivo using xenograft tumor model. GSCs were isolated from the U87 and LN229 cell lines through flow cytometry and the stemness characteristics were verified by immunofluorescence staining. The sphere-forming ability of GSCs was examined using the stem cell sphere formation assay. Bioinformatics methods were conducted to identified the potential downstream target genes of MAD2L2, followed by in vitro experimental validation. Furthermore, potential upstream transcription factors that regulate MAD2L2 expression were confirmed through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. Results The MAD2L2 exhibited high expression in glioblastoma samples and showed significant correlation with patient prognosis. In vitro and in vivo experiments confirmed that silencing of MAD2L2 led to decreased proliferation, invasion, and migration capabilities of glioblastoma cells, while decreasing stemness characteristics of glioblastoma stem cells. Conversely, overexpression of MAD2L2 enhanced these malignant behaviors. Further investigation revealed that MYC proto-oncogene (c-MYC) mediated the functional role of MAD2L2 in glioblastoma, which was further validated through a rescue experiment. Moreover, using dual-luciferase reporter gene assays and ChIP assays determined that the upstream transcription factor E2F-1 regulated the expression of MAD2L2. Conclusion Our study elucidated the role of MAD2L2 in maintaining glioblastoma stemness and promoting malignant behaviors through the regulation of c-MYC, suggesting its potential as a therapeutic target.

Modelling Glioma Stem Cell-mediated Tumorigenesis Using Zebrafish PDX Systems

“…Seed the cells at a density of 1 x 10 4 cells/well and 100uL of HBTC+ infection media or U251-MG infection media/well.…”

“…Glioma stem cells (GSCs), a sub-population of immature neural precursor cells within the GBM mass, resemble normal neural stem cells in their ability of self-renew and differentiate into multiple lineages. GSCs are resistant to conventional therapies and cause tumour recurrence leading to relapse following standard of care therapy 4 . Studying GSCs in vivo can aid in the discovery of unique molecular and biological characteristics that can be targeted to improve the treatment of GBM.…”

Overexpression of EZH2 is associated with clinicopathological parameters and poor prognosis in gliomas

“…Furthermore, multivariate Cox regression analysis con rmed that EZH2 expression, age, and WHO grade were independent risk factors for the survival of glioma patients (Table 3). Abbreviations: CI, con dence interval; HR, hazard ratio Discussion Gliomas are the most common primary central nervous system tumors [29][30][31], with glioblastoma accounting for 60%-75% of cases [32]. Despite the development of combined surgery with locally fractionated radiotherapy and adjuvant temozolomide as the standard rst-line treatment, the 5-year survival rate for glioblastoma patients remains low at 6.8% [33,34].…”