过表达miR-34a增加血肿瘤屏障通透性机制的研究

目的探讨过表达miR-34a增加血肿瘤屏障通透性的机制。方法将miR-34a模拟物转染至培养的人脑微血管内皮细胞NKIM-6,采用实时荧光定量PCR方法检测miR-34a的表达。用miR-34a模拟物转染的NKIM-6细胞和U87胶质瘤细胞建立体外血肿瘤屏障模型,跨内皮电阻测量系统检测血肿瘤屏障跨内皮阻抗值的变化;western blot和免疫荧光法检测体外血肿瘤屏障NKIM-6细胞中,紧密连接相关蛋白ZO-1和claudin-5的表达。结果经miR-34a模拟物转染后,NKIM-6细胞中miR-34a的表达水平显著升高;血肿瘤屏障跨内皮阻抗值显著下降;体外血肿瘤屏障NKIM-6细胞中紧密连接相关蛋白ZO-1和claudin-5的表达水平分别显著降低,在细胞膜上呈不连续分布。结论过表达miR-34a显著增加血肿瘤屏障的通透性,其机制之一可能与降低紧密连接相关蛋白相关。

Overexpression of miR-34a increases blood-tumor barrier permeability

Objective To investigate the mechanism of increase of the blood-tumor barrier(BTB) permeability by overexpressed miR-34a.Methods After transfecting human brain capillary endothelial cell line NKIM-6 with miR34a mimic,real-time PCR analysis was used to detect the expressing level of miR-34a.The NKIM-6 cells overexpressing miR-34a and human U87 glioma cells were used to establish the BTB model in vitro.Transendothelial electrical resistance(TEER) of BTB was measured and the expressing levels of tight junction associated proteins ZO-1 and claudin-5 in NKIM-6 cells were detected by western blot and immunofluorescence staining.Results After transfection with miR-34a mimic,the expressing level of miR-34a in NKIM-6 cells raised up markedly,the TEER of BTB decreased significantly.In the NKIM-6 cells of BTB in vitro,the expressing levels of tight junction associated proteins ZO-1 and claudin-5 were reduced significantly,ZO-1 and claudin-5 positive products showed a discontinuous localization on the membrane of NKIM-6 cells.Conclusion Overexpression of miR-34a increased the BTB permeability significantly,and the down-regulated expression of tight junction associated proteins might be one of the related mechanisms.