U87细胞分泌外囊泡中HOTAIR刺激胶质瘤血管新生的机制研究  

Research on the Mechanism of U87-derived Extracellular Vesicles Mediating HOTAIR Induced Angiogenesis

作　　者：马鑫[1] 濮剑辰 李婷[1] 许颖 范春雷[1] 田男[1] 

机构地区：[1]浙江中医药大学生命科学学院,杭州310053

出　　处：《浙江中医药大学学报》2017年第12期993-999,1006,共8页Journal of Zhejiang Chinese Medical University

基　　金：浙江省新苗人才计划项目(2016R410037)~~

摘　　要：[目的]研究人脑胶质瘤细胞U87分泌的细胞外囊泡(extracellular vesicles,EVs)中HOX转录反义RNA(HOX transcript antisense intergenic RNA,HOTAIR)对人脑胶质瘤血管生成的作用及可能的分子机制。[方法]将U87细胞分为4组:si GFP组、si-NC组、si HOTAIR组和回复组。脂质体法转染U87细胞,收集转染后的细胞上清液,与人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMVEC)共培养。荧光定量PCR检测U87细胞的转染效率。噻唑蓝(methylthiazolyldiphenyl-tetrazolium bromide,MTT)染色、克隆形成、细胞迁移及成管实验检测转染后细胞培养上清液中HOTAIR基因对HBMVEC增殖、迁移和成管能力的影响。采用RNase和Triton X-100处理U87细胞培养上清液,设空白组、RNase组、Triton X-100组、RNase和Triton X-100联合组,试剂盒提取细胞培养上清液中总RNA,荧光定量PCR检测各组上清液HOTAIR基因含量。[结果]与si-NC组比较,si HOTAIR组的HOTAIR表达明显降低(P<0.05);回复组与si HOTAIR组相比,HOTAIR表达明显升高(P<0.01)。与si HOTAIR组U87细胞培养上清液共培养,HBMVEC增殖、形成克隆数量、迁移能力以及成管能力均明显降低(P<0.01)。与空白组相比,RNase组、Triton X-100组U87细胞上清液中HOTAIR基因表达量无明显差异(P>0.05),而RNase和Triton X-100联合组的HOTAIR基因表达量显著降低(P<0.01)。[结论]沉默HOTAIR基因可以抑制U87细胞促血管生成活性,U87细胞可能通过分泌到EVs中的HOTAIR分子参与胶质瘤血管生成调控。[Objective] To explore the effects and potential molecular mechanism of HOTAIR on angiogenesis of humans glioma in extracellular vesicles derived by U87 cells. [Methods] Human glioma cell line U87 cells were divided into four groups： si GFP group, si RNA negative group（si-NC group）,si HOTAIR group and rescue group. HOTAIR was transfected into U87 cells using liposomal technique, then the transfected supernatant was collected for co-culture of human brain microvascular endothelial cells HBMVEC. Real-time quantitative RT-PCR was used to detect the expression levels of HOTAIR gene, and then measured the influence of HOTAIR in supernatant on proliferation, migration and tube formation of HBMVEC by MTT assay,colony formation assay, cell migration assay and tube formation assay. To confirm that HOTAIR was present in the supernatant of the extracellular vesicles, the U87 cell culture supernatant was treated with RNase and Triton X-100, the culture supernatant was divided into four groups： the blank group, RNase group, Triton X-100 group, RNase and Triton X-100 combined group. The kit was used to extract total RNA of extracellular vesicles derived by U87 cells, real-time quantitative RT-PCR was used to detect the expression levels of HOTAIR gene. [Results] Compared with the si-NC group, the expression level of HOTAIR gene of si HOTAIR group was significantly decreased（P〈0.05）, but the rescue group was increased（P〈0.01）; the ability of HBMVEC on proliferation, colony formation, migration and tube formation was obviously reduced（P〈0.05）. In addition, the expression levels of HOTAIR gene in RNase group and Triton X-100 group was not significantly different from that in blank group（P〈0.05）, but the expression levels of HOTAIR gene was significantly decreased in RNase and Triton X-100 group（P〈0.01）. [Conclusion] Knockdown HOTAIR could inhibit pro-angiogenic activity of U87 cells, HOTAIR molecular might participate in regulating the process of angiogenesis by the extracellular vesicles whi

关 键 词：人脑胶质瘤 HOTAIR 血管生成 U87 HBMVEC EVS 分子机制 基因 

分 类 号：R331[医药卫生—人体生理学]