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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。
Objective To investigate the mechanism of overexpression of miR-34a in increasing the permeability of hematological tumor barrier. Methods miR-34a mimics were transfected into cultured human brain microvascular endothelial cells NKIM-6, and the expression of miR-34a was detected by real-time fluorescence quantitative PCR. The in vitro hematological tumor barrier model was established by transfecting NKIM-6 cells and U87 glioma cells with miR-34a mimics. The trans-endothelial resistance assay system was used to detect the trans-endothelial impedance changes of the tumor. Western blot and immunofluorescence assay were used to detect in vitro Tumor-associated NKIM-6 cells are closely linked to the expression of ZO-1 and claudin-5. Results The expression of miR-34a in NKIM-6 cells was significantly increased after transfection with miR-34a mimics; the trans-endothelial impedance was significantly decreased in the blood-tumor barrier; the tight junction-associated protein ZO -1 and claudin-5 expression levels were significantly reduced, respectively, in the cell membrane was discontinuous distribution. Conclusion Overexpression of miR-34a significantly increases the permeability of the hematological tumor barrier, and one of the mechanisms may be related to the reduction of tight junction-related proteins.