Kuehbacher Angelika, Urbich Carmen, Zeiher Andreas M, Dimmeler Stefanie
Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Circ Res. 2007 Jul 6;101(1):59-68. doi: 10.1161/CIRCRESAHA.107.153916. Epub 2007 May 31.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to the cellular transcript leading to translational repression or degradation of the target mRNA. Dicer and Drosha are the miRNA processing enzymes that are required for the maturation of miRNAs. Here, we investigated the role of Dicer and Drosha for angiogenesis. Endothelial cells were transfected with siRNA against Dicer and Drosha to inhibit miRNA biogenesis. Genetic silencing of Dicer and Drosha significantly reduced capillary sprouting of endothelial cells and tube forming activity. Migration of endothelial cells was significantly decreased in Dicer siRNA-transfected cells, whereas Drosha siRNA had no effect. Silencing of Dicer but not of Drosha reduced angiogenesis in vivo. Next, we attempted to identify miRNAs expressed in endothelial cells. A screening analysis of 168 human miRNAs using real-time PCR revealed that members of the let-7 family, mir-21, mir-126, mir-221, and mir-222 are highly expressed in endothelial cells. Dicer and Drosha siRNA reduced lef-7f and mir-27b expression. Inhibitors against let-7f and mir-27b also reduced sprout formation indicating that let-7f and mir-27b promote angiogenesis by targeting antiangiogenic genes. In silico analysis of predicted targets for let-7 cluster identified the endogenous angiogenesis inhibitor thrombospondin-1. Indeed, Dicer and Drosha siRNA significantly increased the expression of thrombospondin-1. Taken together, transient reduction of the miRNA-regulating enzyme Dicer impairs angiogenesis in vitro and in vivo, whereas Drosha siRNA induced a minor antiangiogenic effect in vitro and was not effective in vivo. The let-7 family and mir-27b appear to be attractive targets for modulating angiogenesis.
微小RNA（miRNA）是一类小的非编码RNA，通过与细胞转录本结合来调节基因表达，从而导致靶mRNA的翻译抑制或降解。Dicer和Drosha是miRNA成熟所需的加工酶。在此，我们研究了Dicer和Drosha在血管生成中的作用。用针对Dicer和Drosha的小干扰RNA（siRNA）转染内皮细胞，以抑制miRNA的生物合成。Dicer和Drosha的基因沉默显著降低了内皮细胞的毛细血管芽生和管形成活性。在转染Dicer siRNA的细胞中，内皮细胞的迁移显著减少，而Drosha siRNA则无此作用。Dicer而非Drosha的沉默在体内降低了血管生成。接下来，我们试图鉴定在内皮细胞中表达的miRNA。使用实时聚合酶链反应（PCR）对168种人类miRNA进行的筛选分析表明，let-7家族成员、mir-21、mir-126、mir-221和mir-222在内皮细胞中高度表达。Dicer和Drosha siRNA降低了lef-7f和mir-27b的表达。针对let-7f和mir-27b的抑制剂也减少了芽生形成，表明let-7f和mir-27b通过靶向抗血管生成基因促进血管生成。对let-7簇预测靶标的计算机分析确定了内源性血管生成抑制剂血小板反应蛋白-1。实际上，Dicer和Drosha siRNA显著增加了血小板反应蛋白-1的表达。综上所述，miRNA调节酶Dicer的短暂减少在体外和体内损害血管生成，而Drosha siRNA在体外诱导轻微的抗血管生成作用，在体内则无效。let-7家族和mir-27b似乎是调节血管生成的有吸引力的靶点。
