TET甲基胞嘧啶双加氧酶1调控胶质母细胞瘤免疫微环境的机制研究  

Study on the mechanism of TET methylcytosine dioxygenase 1 regulating the immune microenviron-ment of glioblastoma

作　　者：蒲焯楠 刘金秋 邓宇轩 张小利 吕一帆 杨明煦 郝淑煜[2] 季楠[2] 万虹[1] 冯洁[1] Pu Zhuonan;Liu Jinqiu;Deng Yuxuan;Zhang Xiaoli;Lyu Yifan;Yang Mingxu;Hao Shuyu;Ji Nan;Wan Hong;Feng Jie(Beijing Neurosurgical Institute,Capital Medical University,Beijing 100070,China;Neurosurgery Center,Beijing Tiantan Hospital,Capital Medical University,Beijing 100070,China)

机构地区：[1]首都医科大学,北京市神经外科研究所,北京100070 [2]首都医科大学附属北京天坛医院神经外科学中心,北京100070

出　　处：《中华神经外科杂志》2025年第1期91-98,共8页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(81702455,81872052)。

摘　　要：目的探讨TET甲基胞嘧啶双加氧酶1(TET1)对胶质母细胞瘤(GBM)免疫微环境的影响。方法基于中国脑胶质瘤基因组图谱(CGGA)数据库(504例)、癌症基因组图谱(TCGA)数据库(142例)和首都医科大学附属北京天坛医院神经外科学中心23例GBM患者肿瘤样本RNA测序数据库数据,以TET1表达量的中位数为标准分为TET1高表达组和低表达组(3个数据库对应例数分别为252和252、70和72、12和11例)。筛选3个数据库中TET1高表达组与低表达组间的差异表达基因(DEGs)及其互作网络图中的关键节点基因。采用基因本体论(GO)、京都基因和基因组百科全书(KEGG)对同时存在于3个数据库的DEGs进行富集分析,采用"xCell"分析TET1高表达组与低表达组在3个数据库中免疫细胞浸润及相关评分情况。慢病毒转染GBM细胞株LN18获得TET1干扰RNA(shTET1)组和空载转染(shNC)组,采用实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹(WB)法检测TET1敲低后TET1的表达情况。收集shNC组和shTET1组LN18细胞上清液(即得到相应的GBM细胞条件培养基)后分别与U937巨噬细胞共培养,流式细胞术检测共培养后U937细胞M1、M2型巨噬细胞的占比。采用RT-qPCR检测shNC组和shTET1组LN18细胞中趋化因子2(CCL2)、色氨酸2,3双加氧酶(TDO2)及Toll样受体2(TLR2)的表达情况。结果共筛选出73个DEGs同时存在于上述3个数据库中,其中CCL2、TDO2、TLR2等为DEGs互作网络中的关键节点基因。GO和KEGG富集分析显示,DEGs主要参与GBM细胞的免疫微环境调控相关通路等。"xCell"分析显示,与TET1低表达组相比,在CGGA和TCGA数据库中,TET1高表达组表现出B细胞和巨噬细胞的浸润明显改变,而在23例GBM患者肿瘤样本RNA测序数据库中,TET1高表达组仅表现出巨噬细胞的浸润明显下降。免疫评分结果显示,与TET1低表达组比较,在CGGA和TCGA数据库中,TET1高表达组的免疫评分和微环境评分均降低(均P<0.05)。RT-qPCR结果显Objective To investigate the impact of TET methylcytosine dioxygenase1(TET1)on the immune microenvironment of glioblastoma(GBM).Methods Using RNA-sequencing data from the China Glioma Genome Atlas(CGGA)(504 cases),The Cancer Genome Atlas(TCGA)(142cases),and 23 GBM samples obtained from Beijing Tiantan Hospital,samples were divided into TET1 high and low-expression groups respectively(the corresponding patient numbers for the three databases are 252 and 252,70 and 72,and 12 and 11,respectively).Differentially expressed genes(DEGs)present in all three databases were selected for further analysis.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed.The"xCell"tool was used to assess immune cell infiltration and to calculate immune score.Real-time quantitative PCR(RT-qPCR)and Western Blot(WB)were used to detect TET1 expression in stable TET1-konckdown GBM cell lines by shRNA(shTET1)and those transfecting negative control(shNC).Conditioned media from shNC and shTET1 GBM cells were co-cultured with U937 cell line,and flow cytometric analysis was used for detecting the proportion of M1 and M2 type macrophages polarized from U937,respectively.Additionally,RT-qPCR were used to analysis the expression of chemokine ligand(CCL2),Tryptophan 2,3-Dioxygenase(TDO2),and Toll-like receptor 2(TLR2),in shNC and shTET1 LN18 cell lines.Results A total of 73 DEGs were screened out and co-existed in the above three databases,among which CCL2,TDO2,TLR2,and CD163 were the key nodes in the interaction network.GO and KEGG enrichment analysis showed that 73 DEGs were mainly involved in immune-related pathways.The"xCell"analysis showed that,compared with the TET1 low-expression group,the high-expression group showed a significant changes in B cell and macrophage infiltration in the TCGA and CGGA databases,while it merely showed a significant decrease in macrophage infiltration in the 23 GBM samples.Furthermore,compared with the TET1 low-expression group,immune scores and microenvironment scores w

关 键 词：胶质母细胞瘤 TET甲基胞嘧啶双加氧酶1 免疫细胞浸润 免疫微环境 机制 

分 类 号：R739.41[医药卫生—肿瘤]