Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells.
Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells.
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DOI：
10.1042/bj20101585
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发表时间：
2011-03-15
期刊：
影响因子：
--
通讯作者：
Kalra VK
中科院分区：
文献类型：
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作者：
Patel N;Tahara SM;Malik P;Kalra VK
纤溶酶原激活物抑制剂-1（派-1）是一种重要的生理性纤溶抑制剂。此前，我们报道了胎盘生长因子（PlGF）通过激活人肺微血管内皮细胞（HPMVEC）中的缺氧诱导因子-1 α和激活蛋白-1介导的派-1 mRNA表达转录上调;这导致镰状细胞性贫血（SCA）患者的派-1升高。在此，我们通过检测microRNA在PlGF诱导的派-1 mRNA稳定性中的作用，鉴定了PlGF介导的PAI-1 mRNA在HPMVEC中积累的转录后机制的作用。我们的研究结果表明，miR-30 c和miR-301 a的表达减少，但不响应于PlGF的miR-99 a，其在派-1 mRNA的3′-非翻译区（3′UTR）具有进化上保守的结合位点。抗miR-30 c或抗miR-301 a寡核苷酸的转染导致派-1 mRNA水平增加，其在PlGF刺激下进一步增加。相反，pre-miR-30 c或pre-miR-301 a的过表达导致PlGF诱导的派-1 mRNA和蛋白水平的减弱。使用野生型和突变型3′UTR构建体的荧光素酶报告基因测定证实派-1-3′UTR确实是miR-30 c和miR-301 a的直接靶点。最后，与正常对照组相比，SCA患者的miR-30 c和miR-301 a血浆水平显著下调。这些结果为PlGF诱导PAI-1升高提供了转录后调节机制。
Plasminogen activator inhibitor-1 (PAI-1) is a key physiological inhibitor of fibrinolysis. Previously, we reported placenta growth factor (PlGF) mediated transcriptional upregulation of PAI-1 mRNAexpression via activation of hypoxia-inducible factor-1α and activator protein-1 in human pulmonary microvascular endothelial cells (HPMVEC); which resulted in elevated PAI-1 in humans with sickle cell anemia (SCA). Herein, we identified the role of post-transcriptional mechanism(s) of PlGF-mediated accumulation of PAI-1 mRNA in HPMVEC by examining the role of microRNAs in PlGF-induced PAI-1 mRNA stability. Our results show reduced expression of miR-30c and miR-301a, but not of miR-99a in response to PlGF, which have evolutionarily conserved binding sites in the 3′-untranslated region (3′UTR) of PAI-1 mRNA. Transfection of anti-miR-30c or anti-miR-301a oligonucleotides resulted in increased PAI-1 mRNA levels, which were further increased with PlGF stimulation. Conversely, overexpression of pre-miR-30c or pre-miR-301a resulted in attenuation of PlGF-induced PAI-1 mRNA and protein levels. Luciferase reporter assays using wild-type and mutant 3′UTR constructs confirmed that PAI-1-3′UTR is indeed a direct target of miR-30c and miR-301a. Finally, plasma levels of miR-30c and miR-301a were significantly downregulated in patients with SCA, compared to normal controls. These data provide a post-transcriptional regulatory mechanism of PlGF-inducedPAI-1elevation.