miR-200c调控RhoA基因表达介导RMP7增加血肿瘤屏障通透性机制的研究

发布时间：2018-03-25 01:25

  本文选题：miR-c　切入点：Ras基因家族成员A　出处：《中国医科大学学报》2016年12期

【摘要】：目的研究miR-200c调控Ras基因家族成员A(RhoA)表达介导RMP7增加血肿瘤屏障(BTB)通透性的机制。方法应用real-time PCR检测RMP7作用BTB后人脑微血管内皮细(ECs)miR-200c的表达;应用miR-200c模拟物和miR-200c抑制物分别转染GECs(ECs和U87脑胶质瘤细胞共培养的细胞),测量跨内皮阻抗值(TEER)、辣根过氧化物酶(HRP)渗漏量,分析BTB通透性;应用Western blotting检测RhoA的表达;应用免疫荧光方法观察GECs中RhoA表达和分布;应用双荧光素酶报告基因检测miR-200c转录后水平调控RhoA机制。结果 RMP7作用GECs后,使GECs内源性miR-200c表达显著降低;miR-200c模拟物和miR-200c抑制物成功转染到GECs中;miR-200c模拟物显著抑制RMP7诱导TEER值的降低、HRP的升高;miR-200c模拟物显著减少RhoA的表达,促使RhoA在GECs细胞质和细胞核分布减少;miR-200c模拟物转录后水平负性调控RhoA基因表达。miR-200c抑制物与miR-200c模拟物实验结果相反。结论 miR-200c转录后水平负性调控RhoA表达,介导RMP7增加血肿瘤屏障通透性机制。
[Abstract]:Objective to investigate the mechanism of miR-200c regulating the expression of Ras gene family member, Agnia RhoA, and mediate the increase of RMP7 permeability of BBB. Methods real-time PCR was used to detect the expression of ECsmmiR-200c in human brain microvascular endothelial cells after RMP7 treatment with BTB. MiR-200c mimics and miR-200c inhibitors were used to transfect GECs(ECs and U87 glioma cells, respectively, to measure the transendothelial impedance and horseradish peroxidase (HRP) leakage, to analyze the permeability of BTB, and to detect the expression of RhoA by Western blotting. The expression and distribution of RhoA in GECs were observed by immunofluorescence method, and the RhoA mechanism was detected by double luciferase reporter gene. The expression of endogenous miR-200c in GECs was significantly decreased after transfection of miR-200c inhibitor and mimic of miR-200c into GECs. MiR-200c mimics significantly inhibited the increase of TEER value induced by RMP7. The mimic of RMP7 R-200c significantly reduced the expression of RhoA. The expression of RhoA gene was negatively regulated by RhoA at the posttranscriptional level of GECs mimics. MiR-200c inhibitor was contrary to that of miR-200c mimics. Conclusion miR-200c posttranscriptional level negatively regulates RhoA expression. Mediates RMP7 to increase the permeability of blood tumor barrier.
【作者单位】： 中国医科大学基础医学院神经生物学教研室;
【基金】：国家自然科学基金(81101918;81673028)
【分类号】：R363

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