MIAT过表达载体的构建及其对血肿瘤屏障通透性的影响

目的构建长链非编码RNA心肌梗死相关转录物(MIAT)过表达载体pcDNA3.1-MIAT,并研究其对血肿瘤屏障通透性影响。方法从与脑胶质细胞U87共培养的人脑微血管内皮细胞hCMEC/D3(GECs)提取总RNA,用反转录聚合酶链式反应技术扩增出MIAT基因片段,通过无缝连接技术克隆到pcDNA3.1载体中进行序列分析,转染重组质粒pcDNA3.1-MIAT到GECs中,real-time PCR检测转染效率,应用跨内皮电阻测量系统和辣根过氧化物酶渗漏实验分析血肿瘤屏障通透性的变化。结果成功扩增出MIAT基因,成功构建pcDNA3.1-MIAT表达载体,Blast序列分析与GenBank中NR00349一致。转染pcDNA3.1-MIAT后,96h转染效率最高,过表达MIAT组跨内皮阻抗值最低,辣根过氧化物酶流量最高,血肿瘤屏障通透性最高。结论成功构建pcDNA3.1-MIAT表达载体,过表达MIAT使血肿瘤屏障通透性升高。

Construction of MIAT overexpression vector and its effect on blood tumor barrier permeability

Objective To construct a pcDNA3.1-MIAT expression vector and study its effect on blood tumor barrier(BTB)permeability.Methods Total RNA was extracted from GECs(ECs with U87 glioma cells co-culturing)by TRIZOL reagents.The LncRNA MIAT fragment was amplified by RT-PCR,cloned into pcDNA3.1 vector and for sequence analysis by seamless ligation.The recombinant plasmid pcDNA3.1-MIAT was transfected into GECs,and transfection efficiency was detected by real-time PCR.The permeability of BTB was detected by the value of transendothelial electric resistance(TEER)and horseradish peroxidase(HRP)flux.Results High-quality totalRNA was extracted from human placenta.A fragment of human MIAT gene was amplified by RT-PCR.pcDNA3.1-MIAT vector was constructed successfully,and the result of Blast sequence analysis is in accordance with that of NR_00349 in GenBank.The highest transfection efficiency occurred at 96h.The permeability of BTB increased in MIAT up-expression group.Conclusion The pcDNA3.1-MIAT expression vector was successfully constructed,and overexpression of MIAT increased the permeability of the BTB.