胶质瘤干细胞外泌体微小RNA-574-5p的表达及在肿瘤侵袭中的作用  

Effects of exosomal microRNA-574-5p from tumor stem-like cells on invasion and migration of glioma

作　　者：鲍子戌 胡珊珊 牛朝诗[1,3] Bao Zixu;Hu Shanshan;Niu chaoshi(Department of Neurosurgery,Anhui Provincial Hospital Affiliated to Anhui Medical University,Anhui Province Key Laboratory of Brain Function and Diseases,Anhui Provincial Stereotactic Neurosurgical Institute,Hefei 230001,China)

机构地区：[1]安徽医科大学附属安徽省立医院神经外科,合肥230001 [2]中国科学技术大学附属第一医院神经外科、脑功能与脑疾病安徽省重点实验室、安徽省脑立体定向神经外科研究所 [3]合肥中国科学技术大学附属第一医院神经外科、脑功能与脑疾病安徽省重点实验室、安徽省脑立体定向神经外科研究所

出　　处：《立体定向和功能性神经外科杂志》2021年第4期204-210,共7页Chinese Journal of Stereotactic and Functional Neurosurgery

基　　金：中央引导地方科技创新专项(编号:2017070802D144,2019b07030001)。

摘　　要：目的探讨胶质瘤干细胞(glioma stem-like cells,GSCs)来源的外泌体(exosome)中微小RNA-574-5p(miR-574-5p)的表达水平及其对胶质瘤细胞侵袭能力的影响。方法利用神经细胞球培养法分离恶性胶质瘤细胞系U87来源的胶质瘤干细胞(GSCs),并用免疫荧光染色鉴定其表面标志物。收集胶质瘤干细胞上清液并提取其中的外泌体(GSCs-Exosome),利用深度测序技术分析其中不同微小RNA的表达水平。实时荧光定量PCR(RT-qPCR)验证测序结果。利用靶向miR-574-5p的特异性模拟物及抑制剂转染胶质瘤细胞调控miR-574-5p的表达水平。应用细胞划痕、Transwell实验分别检测GSCs-Exosome及不同miR-574-5p含量对胶质瘤细胞侵袭及迁移能力的影响。结果肿瘤球于神经干细胞培养基中呈悬浮生长,免疫荧光染色证实其特异性表达肿瘤干细胞标志物CD133及神经细胞标志物Nestin。胶质瘤干细胞来源的外泌体中miR-574-5p的含量明显高于人脑正常胶质细胞来源的外泌体,RT-qPCR验证测序结果可靠(均P<0.001)。与对照组相比,GSCs-Exosomes处理的胶质瘤细胞侵袭及迁移能力明显增强(均P<0.05)。此外,过表达miR-574-5p同样明显促进肿瘤细胞侵袭及迁移能力,而抑制miR-574-5p则抑制其侵袭能力(均P<0.05)。结论微小RNA-574-5p在胶质瘤干细胞来源的外泌体中高表达,胶质瘤干细胞外泌体及miR-574-5p均明显促进胶质瘤细胞侵袭及迁移能力。因此,外泌体中微小RNA-574-5p可能作为胶质瘤治疗的新靶点。Objective To explore the expression level of microRNA-574-5 p contained in glioma stem-like cells(GSCs)derived exosomes and its effect on the invasion and migration of glioma cells.Methods Glioma stem-like cells(GSCs)were isolated from U87 cell lines with neural sphere culture method and identified by immunofluorescence assays.The serum-free neural culture medium was collected to isolate GSCs-derived exosomes.The different expression pattern of microRNAs in exosomes from GSCs and human normal glia cells(HEB)were detected by deep sequencing technology.The result of deep sequencing was validated with RT-qPCR.Specific mimic and inhibitor were used to regulate the level of miR-574-5 p in glioma cells.Wound healing and Transwell assay were performed the effect of GSCs-exosomes and miR-574-5 p on the invasion and migration of glioma cells.Results The isolated glioma spheres were suspended in the neural culture medium.The surface marker of tumor stem cells(CD133)and neural cells(Nestin)were identified by immunofluorescence staining in glioma spheres.The result of deep sequencing indicated that the level of miR-574-5 p was significantly up-regulated in GSCs-derived exosomes compared to exosomes from HEB,which was also validated by RT-qPCR(P<0.001).Besides,the invasion and migration of glioma cells treated with GSCs-derived tyexosomes were significantly enhanced compared to normal control(P<0.05).Overexpression of miR-574-5 p also promoted the invasion of glioma cells,while inhibition of miR-574-5 p reduced the aggressiveness(P<0.05).Conclusion microRNA-574-5 p was significantly up-regulated in GSCs-derived exosomes.Both GSCs-derived exosomes and miR-574-5 p were able to promote the aggressiveness of glioma cells,which suggested that exosomal miR-574-5 p could be the novel therapeutic target for glioma treatment.

关 键 词：外泌体 神经胶质瘤 胶质瘤干细胞 微小RNA miR-574-5p 肿瘤侵袭 

分 类 号：R739.11[医药卫生—肿瘤]