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Abstract：
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.
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-2\'-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.
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.
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.
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-2\'-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.
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.
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.
摘要：
背景：胶质母细胞瘤，最常见的原发性恶性肿瘤,与预后不良有关。胶质母细胞瘤细胞表现出高增殖和侵袭特性，和胶质母细胞瘤干细胞（GSCs）已被证明在胶质母细胞瘤细胞的恶性行为中起关键作用。本研究旨在探讨GSCs维持和恶性进展的分子机制。
方法：基于公共数据库的数据进行生物信息学分析，以探索有丝分裂阻滞缺陷2样2（MAD2L2）的表达谱及其在神经胶质瘤中的潜在功能。通过细胞活力测定（CCK8）评估MAD2L2对胶质母细胞瘤细胞行为的影响，集落形成试验,5-乙炔基-2'-脱氧尿苷(EDU)掺入测定，划痕试验,和transwell迁移/入侵测定。使用异种移植肿瘤模型在体内进一步验证来自体外实验的发现。通过流式细胞术从U87和LN229细胞系中分离GSCs，并通过免疫荧光染色验证其干性特征。使用干细胞球形成测定检查GSC的球形成能力。采用生物信息学方法鉴定MAD2L2的潜在下游靶基因,并进行体外实验验证。此外，调节MAD2L2表达的潜在上游转录因子通过染色质免疫沉淀(ChIP)和双荧光素酶报告基因测定得到证实.
结果：MAD2L2在胶质母细胞瘤样本中呈高表达，与患者预后显著相关。体外和体内实验证实，沉默MAD2L2导致增殖减少，入侵,和胶质母细胞瘤细胞的迁移能力，同时降低胶质母细胞瘤干细胞的干性特征。相反，MAD2L2的过度表达增强了这些恶性行为。进一步研究发现MYC原癌基因(c-MYC)介导MAD2L2在胶质母细胞瘤中的功能作用,通过救援实验进一步验证。此外，使用双荧光素酶报告基因测定和ChIP测定确定上游转录因子E2F-1调节MAD2L2的表达。
结论：我们的研究阐明了MAD2L2通过调节c-MYC在维持胶质母细胞瘤干性和促进恶性行为中的作用，表明其作为治疗靶点的潜力。
方法：基于公共数据库的数据进行生物信息学分析，以探索有丝分裂阻滞缺陷2样2（MAD2L2）的表达谱及其在神经胶质瘤中的潜在功能。通过细胞活力测定（CCK8）评估MAD2L2对胶质母细胞瘤细胞行为的影响，集落形成试验,5-乙炔基-2'-脱氧尿苷(EDU)掺入测定，划痕试验,和transwell迁移/入侵测定。使用异种移植肿瘤模型在体内进一步验证来自体外实验的发现。通过流式细胞术从U87和LN229细胞系中分离GSCs，并通过免疫荧光染色验证其干性特征。使用干细胞球形成测定检查GSC的球形成能力。采用生物信息学方法鉴定MAD2L2的潜在下游靶基因,并进行体外实验验证。此外，调节MAD2L2表达的潜在上游转录因子通过染色质免疫沉淀(ChIP)和双荧光素酶报告基因测定得到证实.
结果：MAD2L2在胶质母细胞瘤样本中呈高表达，与患者预后显著相关。体外和体内实验证实，沉默MAD2L2导致增殖减少，入侵,和胶质母细胞瘤细胞的迁移能力，同时降低胶质母细胞瘤干细胞的干性特征。相反，MAD2L2的过度表达增强了这些恶性行为。进一步研究发现MYC原癌基因(c-MYC)介导MAD2L2在胶质母细胞瘤中的功能作用,通过救援实验进一步验证。此外，使用双荧光素酶报告基因测定和ChIP测定确定上游转录因子E2F-1调节MAD2L2的表达。
结论：我们的研究阐明了MAD2L2通过调节c-MYC在维持胶质母细胞瘤干性和促进恶性行为中的作用，表明其作为治疗靶点的潜力。
