MCM5促进胶质母细胞瘤恶性进展及其机制研究  

Role of microchromosome maintenance protein 5 in promoting malignant progression and its mechanism in glioblastoma

作　　者：冯如玥 谢琳[1] 欧阳乐平[1] 何明亮[1] 刘家豪[1] 刘安民[1] Feng Ruyue;Xie Lin;Ouyang Leping;He Mingliang;Liu Jiahao;Liu Anmin(Department of Neurosurgery,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120,China)

机构地区：[1]中山大学孙逸仙纪念医院神经外科,广州510120

出　　处：《中华神经医学杂志》2022年第3期217-225,共9页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(8167102120)。

摘　　要：目的探究微小染色体维持蛋白5(MCM5)对胶质母细胞瘤的恶性进展促进作用及机制。方法(1)收集中山大学孙逸仙纪念医院神经外科自2020年9月至2021年9月术中切除的3例非肿瘤患者脑组织与3例Ⅳ级胶质母细胞瘤组织进行质谱检测及蛋白质组学定量分析,筛选差异表达的蛋白并进行功能富集分析。(2)在蛋白质组学的基础上筛选出在胶质母细胞瘤中高表达的目标蛋白MCM5,利用癌症基因组图谱(TCGA)数据库验证其在胶质母细胞瘤中表达情况,并在收集到的临床标本中进行mRNA水平的进一步验证。(3)通过siRNA转染将U251细胞分为阴性对照组、敲低组-1(si-1组)、敲低组-2(si-2组),采用CCK-8实验、平板克隆形成实验、EdU染色、Transwell实验及流式细胞术检测MCM5对胶质母细胞瘤恶性表型的调控作用。(4)利用TCGA数据库中胶质瘤患者的转录组数据,通过GSEA算法探究MCM5调控胶质母细胞瘤恶性进程可能的分子机制。结果(1)在胶质母细胞瘤临床样本中筛选出表达上调的蛋白322种,表达下调的蛋白94种,其中MCM5在3份胶质母细胞瘤样本中均呈现为高表达。(2)基于TCGA数据库163例胶质母细胞瘤和207例非肿瘤脑组织的分析显示MCM5在胶质母细胞瘤中表达上调,差异有统计学意义(t=3.340,P=0.001);针对临床收集的3份胶质母细胞瘤组织和3份非肿瘤脑组织的实时定量PCR(RT-qPCR)实验结果同样显示MCM5在胶质母细胞瘤中表达增高,差异有统计学意义(t=3.876,P<0.001)。(3)与阴性对照组比较,si-1组、si-2组细胞MCM5 mRNA及蛋白表达均显著下降,接种后第5天的增殖率显著降低,细胞克隆数量显著减少,EdU阳性细胞比例显著降低,G1期细胞比例显著上升,迁移能力明显受损,差异均有统计学意义(P<0.05)。(4)GSEA分析结果显示,MCM5高表达组mRNA在DNA损伤修复、E2F靶基因、MYC靶基因、上皮-间质转化(EMT)、白细胞介素6-Janus激酶-信号转导与Objective To investigate the role of microchromosome maintenance protein 5(MCM5)in promoting malignant progression and its mechanism in glioblastoma.Methods(1)Three freshly excised brain tissues from non-tumor patients and 3 grading IV glioblastoma tissues were collected in our hospital from September 2020 to September 2021;mass spectrometry and quantitative proteomic analysis were performed to screen differentially expressed proteins for functional enrichment analysis.(2)The target protein MCM5,which was highly expressed in glioblastoma,was screened on the basis of proteomics,and its expression in glioblastoma was verified using The Cancer Genome Atlas(TCGA)database and further validated at the mRNA level in the collected clinical specimens.(3)U251 cells were divided into negative control group,knockdown group-1(si-1 group)and knockdown group-2(si-2 group)by siNRA transfection.The regulation role of MCM5 in malignant phenotype of glioblastoma was detected by CCK-8 assay,clone formation assay,5-ethynyl-2'-deoxyuridine(EdU)staining,Transwell invasion assay and flow cytometry.(4)The transcriptome data of glioma patients from TCGA database were used to explore the possible molecular mechanisms of MCM5 regulation in the malignant process of glioblastoma by gene set enrichment analysis(GSEA)algorithm.Results(1)In clinical samples of glioblastoma,322 up-regulated proteins and 94 down-regulated proteins were screened out;MCM5 was highly expressed in these 3 glioblastoma samples.(2)Based on TCGA database,results of 163 patients with glioblastoma and 207 patients with non-tumor brain tissues showed that MCM5 expression was statistically up-regulated in glioblastoma(t=3.340,P<0.001).Real-time quantitative PCR results of 3 glioblastoma tissues and 3 non-tumor brain tissues clinically collected in our hospital also indicated that significantly increased MCM5 expression in glioblastoma was noted as compared with that in the non-tumor brain tissues(t=3.876,P<0.001).(3)As compared with the negative control group,the si-1 and si

关 键 词：微小染色体维持蛋白5 胶质母细胞瘤 细胞增殖 细胞周期 

分 类 号：R739.4[医药卫生—肿瘤]