小非编码RNA-376b-3p在恶性胶质瘤患者血清外泌体中的表达及抗血管形成的作用机制  

Study on the expression of non-coding microRNA-376b-3p in serum exosomes of patients with malignant glioma and the mechanism of anti-angiogenesis

作　　者：姜军[1] 王顺[2] 孟庆虎[1] 于锐[1] 魏胜程[1] 王骥[1] 曲春城[1] 王成伟[1] Jiang Jun;Wang Shun;Meng Qinghu;Yu Rui;Wei Shengcheng;Wang Ji;Qu Chuncheng;Wang Chengwei(Department of Neurosurgery,the Second Hospital of Shandong University,Jinan 250033,China;Department of Laboratory Medicine Center,the Second Hospital of Shandong University,Jinan 250033,China)

机构地区：[1]山东大学第二医院神经外科,济南250033 [2]山东大学第二医院检验医学中心,济南250033

出　　处：《中华医学杂志》2020年第21期1634-1639,共6页National Medical Journal of China

基　　金：山东省重点研发计划(公益性科技攻关类)(2019GSF108147);山东省自然科学基金(青年基金)(ZR2019PH105);山东大学第二医院院管项目(Y2015010023)。

摘　　要：目的筛选恶性胶质瘤患者血清外泌体中差异的微小RNA(microRNA),探讨小非编码RNA-376b-3p(miR-376b-3p)对胶质瘤细胞的增殖、侵袭和肿瘤血管生成拟态的影响,并验证其对同源框蛋白D10(HOXD10)的靶向作用。方法通过HiSeq/MiSeq高通量测序技术筛选恶性胶质瘤患者血清外泌体中差异的microRNA表达谱及其靶基因和作用途径。小样本评估候选microRNA在高级别胶质瘤血清外泌体中的表达。采用U87胶质瘤细胞株分别转染对照抑制物、miR-376b-3p抑制物、对照模拟物和miR-376b-3p模拟物后,通过四甲基偶氮唑盐比色法(MTT)实验、细胞迁移实验、血管形成拟态实验检测miR-376b-3p对胶质瘤细胞的增殖率、细胞侵袭能力和肿瘤血管生成拟态的影响。检测HOXD10的信使RNA(mRNA)和蛋白表达水平,评估miR-376b-3p对HOXD10的调控作用。结果测序筛选到高级别胶质瘤血清外泌体与正常对照之间的差异microRNA个数为144个。在京都基因和基因组百科全书(KEGG)数据库富集到局灶性黏附、肿瘤蛋白多糖等参与胶质瘤调节的通路。小样本验证发现miR-376b-3p在高级别胶质瘤中下调(P<0.01),对高级别胶质瘤的诊断的曲线下面积为0.85(P<0.01)。MTT实验显示转染后48~96 h,miR-376b-3p抑制物组增殖能力高于对照组,转染后48、72 h时,miR-376b-3p模拟物组增殖能力低于对照组。Transwell迁移实验显示,miR-376b-3p抑制物组穿膜细胞数量高于对照抑制物组,miR-376b-3p模拟物组穿膜细胞数量低于对照模拟物组。miR-376b-3p模拟物组的血管形成数低于对照模拟物组。miR-376b-3p抑制物下调了HOXD10的mRNA和蛋白的表达水平,miR-376b-3p模拟物上调了HOXD10的mRNA和蛋白的表达水平。结论miR-376b-3p在高级别胶质瘤患者血清外泌体中下调,而上调miR-376b-3p后能够降低胶质瘤细胞的增殖和侵袭能力,抑制血管形成拟态的形成,同时能增加HOXD10的表达,有望同时抑制两种形式�Objective To screen the different microRNAs in the serum exosomes of patients with malignant glioma,to explore the effect of non-coding microRNA-376b-3p(miR-376b-3p)on the proliferation,invasion and tumor vasculogenic mimicry of glioma cells,and to verify its targeting effect on HOXD10.Methods HiSeq/MiSeq high-throughput sequencing was used to screen the different microRNA expression profiles,target genes and action pathways in the serum exosomes of patients with malignant glioma.Samples were used to evaluate the expression of candidate microRNAs in serum exosomes of high-grade gliomas.The effects of miR-376b-3p on the proliferation,invasion and angiogenesis of glioma cells were detected by MTT assay,Transwell migration assay and Matrigel vasculogenic mimicry assay.The mRNA and protein expression of HOXD10 were detected to evaluate the regulatory effect of miR-376b-3p on it.Results There were 144 different expression microRNAs in the serum exosomes between malignant glioma and the normal control.Focal adhesion and tumor protein polysaccharides were involved in the regulation of glioma enriched by KEGG(Kyoto Encyclopedia of Genes and Genomes).MiR-376b-3p was down regulated in malignant glioma,and AUC of malignant glioma was 0.85(P<0.01).MTT test showed that the proliferation ability of miR-376b-3p inhibitor group was higher than that of the control group,and that of miR-376b-3p mimic group was lower than that of the control group.Transwell migration test showed that the number of transmembrane cells in miR-376b-3p inhibitor group was higher than that in NC inhibitor group,and the number of transmembrane cells in miR-376b-3p mimic group was lower than that in NC mimic group.The number of tubes of vasculogenic mimicry in miR-376b-3p mimic group was lower than that in NC mimic group.MiR-376b-3p inhibitor decreased the expression level of HOXD10 mRNA and protein,and miR-376b-3p mimic increased the expression level of HOXD10 mRNA and protein.Conclusions MiR-376b-3p is down-regulated in the serum exosomes of malignant g

关 键 词：神经胶质瘤 微小RNA 外泌体 

分 类 号：R73[医药卫生—肿瘤]