上调微小RNA-1224-5p表达对人肝癌血管内皮细胞生物学行为的影响  

The effects of up-regulating the expression of microRNA-1224-5p on hepatocellular carcinoma tumor endothelial cells

作　　者：陈麒钧 胡超[1] 沈世强[1] 崔忠惠[1] 闫瑞承[1] 夸蒋 

机构地区：[1]武汉大学人民医院肝胆外科,430060

出　　处：《中华实验外科杂志》2016年第10期2282-2286,共5页Chinese Journal of Experimental Surgery

基　　金：武汉市科技攻关计划项目（201260523178-2）

摘　　要：目的 观察微小RNA-1224-5p（miR-1224-5p）对人肝癌血管内皮细胞（TEC）生物学行为的影响.方法 将合成的miR-1224-5p模拟物（mimic）通过脂质体2000转入TEC细胞中.实验分为3组：（1）上调组（miR-1224-5p mimic,MU组）;（2）阴性对照组（NC组）;（3）空白对照组（CON组）.转染后通过实时定量反转录聚合酶链反应（RT-qPCR）法检测3组细胞中miR-1224-5p的表达量来验证转染是否成功.分别用噻唑蓝（MTT）法检测细胞增殖,流式细胞仪检测细胞的凋亡及细胞周期,Ttranswell迁移和侵袭实验检测3组细胞迁移侵袭能力的变化、血管形成实验检测3组TEC细胞的血管形成能力.结果 RT-qPCR检测结果显示MU组中miR-1224-5p的表达量（2-△△Ct=3.27±0.15）显著高于NC组（2-△△Ct=1.08±0.11）和CON组（2-△△Ct=1.00）,差异有统计学意义（P＜0.01）.MTT结果显示MU组细胞在转染miRNA-1224-5p mimic后的第24、48、72、96小时4个时间点均表现出吸光度值降低,与NC组及CON组比较差异有统计学意义（P＜0.01）.流式细胞仪检测凋亡结果显示CON、NC、MU组凋亡率分别为（8.73±0.64）％、（9.51±0.56）％、（19.29±0.95）％,MU组细胞凋亡率比其他两组明显增加,差异有统计学意义（P＜0.01）.而细胞周期检测结果显示CON、NC、MU组3组细胞中G1期细胞比例分别为（50.7±1.3）％、（51.7±1.9）％、（73.3±2.0）％;S期细胞的比例为（31.4±2.7）％、（29.1±1.6）％、（23.0±3.0）％;MU组细胞中G1期细胞比其他两组显著增加,S期细胞比其他两组显著减少,差异有统计学意义（P＜0.01或P＜0.05）.Transwell细胞迁移实验结果显示CON、NC、MU 3组细胞中从Ttranswell上室迁移到下室的细胞数分别为（77.7±2.5）、（79.2±3.5）、（51.0±3.6）个,与CON组和NC组比较,MU组迁移的细胞数明显下降,差异有统计学意义（P＜0.01）.Transwell细胞侵袭实验结果显示CON、NC、MU 3组细胞中穿Objective To investigate the effect of microRNA-1224-5p （miR-1224-5p） on tumor endothelial cells （TECs） of human hepatocellular carcinoma （HCC）.Methods Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000.TECs were divided into 3 groups：a control （CON） group consisted of TECs without transfection,a negative control （NC） group consisted of TECs transfected with negative control oligonucleotides and Green Fluorescent Protein （GFP）,and a micro-up （MU） group consisted of TECs transfected with miR-1224-5p mimic and GFP.The expression of miR-1224-5p was quantified via quantitative reverse transcription polymerase chain reaction （RT-qPCR）.The proliferation of TECs was detected using MTF （Thiazolyl Blue Tetrazolium Bromide） assay.Apoptosis and cell cycle were detected via flow cytometry.The migration and invasion of TECs were detected using Ttranswell assay.The ability of tube fromation of TECs was detected using tube formation assay.RESULTS Oligonucleotides were successfully transduced into TECs,and specifically upregulated the expression of miR-1224-5p.Results The results of RT-qPCR showed that the expression of miR-1224-5p was significantly increased in the MU group （2-△△Ct =3.27 ± 0.15） compared with the CON group （2-△△Ct =1.00） and the NC group （2-△△Ct=1.08 ±0.11） （P 〈0.01）.The results of MTI assay showed that the cell proliferation was all significantly inhibited in the MU group for the 4 time points compared with the CON group and the NC group （P 〈0.01）.The results of flow cytometry showed that the apoptosis was siguificantly increased in the MU group （19.29 ± 0.95）% compared with the CON group （8.73 ±0.64）% and the NC group （9.51 ±0.56）% （P〈0.01）.The results of cell cycle detection showed that percentage of G1 phase cells in the MU group （73.3 ± 2.0） % was significantly higher than the CON group （50.7 1.3） % and the NC group （51.7 ± 1.9） % and the percentage of

关 键 词：肝细胞肝癌 肝癌血管内皮细胞 微小RNA-1224—5p 

分 类 号：R735.7[医药卫生—肿瘤]