中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2016, Vol. 45 ›› Issue (12): 1057–1062.doi: 10.12007/j.issn.0258-4646.2016.12.001

• 论著 •    下一篇

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

马腾,刘丽波,蔺扬,马珺,薛一雪   

  1. 中国医科大学基础医学院神经生物学教研室,沈阳 110122
  • 收稿日期:2016-06-02 出版日期:2016-12-28 发布日期:2016-12-06
  • 通讯作者: 薛一雪 E-mail:xueyxiue888@163.com
  • 作者简介:马腾(1976 -),男,副教授,博士 .
  • 基金资助:
    国家自然科学基金(81101918;81673028)

miR-200c Regulates RMP7-mediated Increases of Blood-tumor Barrier Permeability by Targeting RhoA

MA Teng,LIU Libo,LIN Yang,MA Jun,XUE Yixue   

  1. Department of Neurobiology,College of Basic Medical Science,China Medical University,Shenyang 110122,China
  • Received:2016-06-02 Online:2016-12-28 Published:2016-12-06

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。

关键词: miR-200c, Ras 基因家族成员 A, RMP7, 血肿瘤屏障, 基因表达调控

Abstract: Objective To study the mechanism of miR-200c in regulating RMP7-induced increases of blood-tumor barrier(BTB)permeability by targeting Ras homolog gene family member A(RhoA). Methods Endogenous expression of miR-200c was detected by real-time PCR in human cerebral microvascular endothelial cell line hCMEC/D3(ECs)after RMP7 treatment. miR-200c mimic and miR-200c inhibitor were transfected into GECs(ECs with U87 glioma cells co-culturing),respectively. Transfection efficiency of miR-200c mimic and miR-200c inhibitor were determined by real-time PCR. HRP flux and TEER assays revealed BTB permeability. The protein expression level of RhoA was assessed by Western blotting. The distribution of RhoA was assessed by immunofluorescence microscopy. RhoA luciferase assays were performed using the Dual-Luciferase reporter assay system. Results RMP7 significantly induced a decrease in miR-200c expression in GECs of BTB. miR-200c mimic and miR-200c inhibitor were successfully transfected into GECs. Overexpression of miR-200c inhibited endothelial leakage and restored normal transendothelial electric resistance values. Simultaneously,overexpression of miR-200c significantly reduced the protein expression level of RhoA. In addition,immunofluorescence analysis revealed that the distribution of RhoA in the cytoplasm and nuclei of GECs were decreased in miR-200c mimic group. RhoA was one of the direct targets of miR-200c with the specific binding site being located at the seed sequence. The results of miR-200c silencing were opposite to that of the miR-200c overexpression group. Conclusion miRNA-200c regulated RMP7-induced increases in BTB permeability by targeting RhoA.

Key words: miR-200c, RhoA, RMP7, blood tumor-barrier, gene expression regulation

中图分类号: 

  • R338.2
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