Two Functional MicroRNA-126s Repress a Novel Target Gene p21-Activated Kinase 1 to Regulate Vascular Integrity in Zebrafish
Two Functional MicroRNA-126s Repress a Novel Target Gene p21-Activated Kinase 1 to Regulate Vascular Integrity in Zebrafish
复制标题
两个功能性 microRNA-126 抑制新的靶基因 p21 激活激酶 1，以调节斑马鱼的血管完整性
DOI：
10.1161/circresaha.110.225045
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发表时间：
2011-02-21
影响因子：
20.1
通讯作者：
Jing, Qing
中科院分区：
文献类型：
--
作者：
Zou, Jun;Li, Wen-Qing;Jing, Qing
基本原理：microRNAs（miRNAs）是血管发育和疾病的关键调节因子。目的：研究内皮细胞miR-126在斑马鱼血管发育中的作用，方法与结果：在斑马鱼基因组中发现了miR-126的两个同源物，即miR-126 a（以前文献中称为miR-126）和miR-126 b，成熟序列仅有1个核苷酸差异。体外分析表明，两种前体都能产生成熟的功能性miRNAs。通过北方印迹和定量RT-PCR的表达分析表明，miR-126在受精后12 h显著积累，并且在斑马鱼的内皮细胞中特异性表达。用特定的吗啉代抑制miR-126 a或miR-126 b会导致颅内出血，同时抑制两种miR-126会导致更高比例的胚胎出现明显出血。生物信息学预测显示，miR-126 a/B的靶点部分重叠，但本质上不同。p21激活激酶1（pak 1）是miR-126 a/B的新靶点，pak 1 3'端非翻译区受这两种miRNAs的不同调控。定量RT-PCR、原位杂交和Western blot分析显示，pak 1的表达水平随着miR-126 a/B的过表达而降低。值得注意的是，当miR-126 a/B被敲低时，内皮细胞中的pak 1表达增加。结论：miR-126 a和miR-126 b是两种内皮细胞特异性的功能性miRNAs，它们协同调节斑马鱼血管的完整性，pak 1是miR-126 a/B在血管发育中的重要靶点。(Circ Res. 2011;108：201-209.）
Rationale: MicroRNAs (miRNAs) are key regulators of vascular development and diseases. The function and underlying mechanism of endothelial miRNAs have not been fully defined.Objective: To investigate the role of endothelial miR-126 in zebrafish vascular development.Methods and Results: Two homologs of miR-126, miR-126a (namely miR-126 in previous literature) and miR-126b, with only 1 nucleotide difference in their mature sequences, were identified in zebrafish genome. In vitro analysis showed that both precursors could sufficiently produce mature functional miRNAs. Expression analyses by Northern blot and quantitative RT-PCR showed that both miR-126s accumulated significantly 12 hours after fertilization and were specifically expressed in endothelial cells of zebrafish. Inhibition of miR-126a or miR-126b with specific morpholinos caused cranial hemorrhage, and simultaneous inhibition of both miR-126s resulted in a pronounced hemorrhage in higher percentage of embryos. Bioinformatics prediction showed that the targets of miR-126a/b partially overlapped but essentially differed. p21-activated kinase1 (pak1) was identified as a novel target of miR-126a/b, and pak1 3' untranslated region was differently regulated by these 2 miRNAs. Quantitative RT-PCR, in situ hybridization, and Western blot analyses showed that the level of pak1 was reduced when miR-126a/b were overexpressed. Notably, pak1 expression in endothelial cells was increased when miR-126a/b were knocked down. Furthermore, overexpression of the active form of human pak1 caused cranial hemorrhage, and knockdown pak1 effectively rescued the hemorrhage caused by inhibiting miR-126a/b.Conclusions: Two functional endothelial cell-specific miRNAs, miR-126a and miR-126b, synergistically regulate zebrafish vascular integrity, and pak1 is a critical target of miR-126a/b in vascular development. (Circ Res. 2011;108:201-209.)