miR-223-3p靶向RIPK3调控胶质瘤微环境中巨噬细胞恶性转化的实验研究  

miR-223-3p regulates the malignant transformation of macrophages in the glioma microenvironment by targeting RIPK3

作　　者：王晔 陆朝晖[1,2] 杨海珍[1] 王海洋 陈俊杰 陈延明[2] 董军[2] Wang Ye;Lu Zhaohui;Yang Haizhen;Wang Haiyang;Chen Junjie;Chen Yanming;Dong Jun(Health Management Center,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Department of Neurosurgery,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Department of Neurosurgery,First People′s Hospital,Hangzhou 310003,China;Department of Neurosur-gery,Wuxi Branch of Zhongda Hospital Affiliated to Southeast University,Wuxi 214105,China)

机构地区：[1]苏州大学附属第二医院健康管理中心,苏州215004 [2]苏州大学附属第二医院神经外科,苏州215004 [3]杭州市第一人民医院神经外科,杭州310003 [4]东南大学附属中大医院无锡分院神经外科,无锡214105

出　　处：《中华神经外科杂志》2023年第3期288-295,共8页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金(81602183);江苏省自然科学基金(BK20201172);江苏省重点研发计划(社会发展)项目(BE2021653);无锡市科技计划项目(N20192010)。

摘　　要：目的探讨miRNAs介导的胶质瘤肿瘤微环境(TME)中巨噬细胞(Mφ)的恶性转化及其机制。方法应用集落刺激因子诱导表达绿色荧光蛋白(GFP)的小鼠骨髓来源细胞(BMDCs)分化,获得Mφ-GFP。将胶质瘤干细胞(GSCs)与Mφ-GFP体外共培养,筛选具备无限增殖潜能的Mφ-GFP,即恶变巨噬细胞(t-Mφ)。通过miRNAs芯片分析获取t-Mφ与正常Mφ的miRNAs差异表达谱。分别比较目标miRNA在中国脑胶质瘤基因组图谱计划(CGGA)不同世界卫生组织(WHO)分级胶质瘤组织、来源于手术切除的10例胶质瘤组织与瘤旁组织、t-Mφ与正常Mφ中的表达水平,并分析目标miRNA表达水平与患者生存期的相关性。采用流式细胞术、实时荧光定量PCR(qRT-PCR)、蛋白质免疫印迹法(WB)及荧光素酶活性实验探讨目标miRNA调控TME中Mφ恶变的分子机制。结果通过建立双色荧光示踪共培养体系,成功获得具备无限增殖潜能的单克隆GFP+细胞,并表达Mφ标志物F4/80和CD11b。miRNAs芯片差异表达谱和qRT-PCR结果证实,与正常Mφ相比,miR-223-3p在t-Mφ中的表达显著升高。临床分析中,胶质瘤组织样本中miR-223-3p的表达水平显著高于配对的瘤旁组织(P<0.01)。对CGGA的分析结果发现,胶质母细胞瘤中的miR-223-3p表达水平显著高于低级别胶质瘤(WHO 2/3级)(P<0.001)。生存分析结果提示,低表达miR-223-3p胶质瘤患者的总体生存期显著优于高表达组(P<0.01)。下调miR-223-3p可促进t-Mφ的凋亡(P<0.01)。生物信息学分析显示,受体相互作用蛋白激酶(RIPK3)基因3′-UTR区存在miR-223-3p潜在的结合位点。荧光素酶活性实验结果显示,与共转染miR-223-3p模拟物对照和RIPK3-WT的细胞相比,共转染miR-223-3p模拟物和RIPK3-WT的t-Mφ细胞荧光强度显著降低(P<0.01)。qRT-PCR和WB实验结果证实,miR-223-3p下调的t-Mφ中RIPK3的表达水平显著高于对照组(P<0.01),而miR-223-3p过表达的t-Mφ中RIPK3的表达水平显著低于对照组(P<0.05)。Objective To investigate the miRNAs-mediated malignant transformation of macro-phages(Mφ)in the glioma tumor microenvironment(TME)and to explore its mechanisms.Methods Bone marrow-derived cells(BMDCs)from green fluorescent protein(GFP)positive mice were obtained,and the BMDCs further directed differentiation into Mφ-GFP.Glioma stem cells(GSCs)expressing red fluorescent protein(GSCs-RFP)were co-cultured with Mφ-GFP in vitro.The monoclonal GFP+cells with indefinite proliferation potential were referred to as malignantly transformed macrophages(t-Mφ).The differential expression profiles of miRNAs between t-Mφand normal Mφwere obtained from the miRNAs microarrays.The expression profiles of the target miRNAs were compared in different WHO grades of glioma tissues from the Chinese Glioma Genome Atlas(CGGA)database between 10 glioma tissues and adjacent normal tissues and between t-Mφand normal Mφ.We also analyzed the correlation between the target miRNAs expression levels and patients′survival time.Flow cytometry,quantitative real-time PCR(qRT-PCR),Western blot,and luciferase report assay were used to analyze the underlying molecular mechanism of target miRNA regulating the malignant transformation of Mφin TME.Results GFP+cells with indefinite proliferation potential were successfully isolated from the dual-color fluorescence co-culture system and expressed Mφmarkers F4/80 and CD11b.Differential expression profiles of miRNAs and qRT-PCR showed that the expression of miR-223-3p was significantly elevated in the t-Mφcompared with normal Mφ.The analysis of CGGA revealed that miR-223-3p expression levels were significantly higher in glioblastomas(WHO grade 4)than those in low-grade gliomas(WHO grade 2/3)(P<0.001).Survival analysis indicated that patients with high expression of miR-223-3p had worse overall survival than those with low expression(P<0.01).Down-regulation of miR-223-3p promoted t-Mφapoptosis(P<0.01).Bioinformatics analysis revealed a potential binding site for miRNA-223-3p in the 3′-UTR regio

关 键 词：神经胶质瘤 肿瘤微环境 巨噬细胞 恶性转化 微RNA-223-3p 

分 类 号：R739.41[医药卫生—肿瘤]