Research Communication miR-200a Modulate HUVECs Viability and Migration Yi-Xuan Li*, Da-Quan Liu*, Chen Zheng*, Shu-Qi Zheng, Min Liu, Xin Li and Hua Tang Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China Summary The posttranscriptional regulation of miRNAs is important for organism development. To investigate the role of miRNAs in angiogenesis, we performed a loss-of-function screening assay in human umbilical vein endothelial cells (HUVECs) and found that knockdown of 7 miRNAs (miR-95a, miR-126, miR-129, miR-137, miR-139, miR-200a, and miR-335) significantly sup- pressed cell viability. As miR-200a was highly expressed in HUVECs, blocking endogenous miR-200a using 2 0 -OMe anti- sense oligonucleotide (ASOs) resulted in a decrease of cell via- bility and migration. Bioinformatics analysis indicates the 3 0 untranslated region (UTR) of thrombospondin-1 (THBS1) has a putative binding site for miR-200a. MiR-200a can directly bind to THBS1 3 0 UTR and negatively regulate THBS1 expression. The identification of endothelial cells (ECs) related miRNA and its target gene may gain new insight into the mechanism of angiogenesis. Ó 2011 IUBMB IUBMB Life, 63(7): 553–559, 2011 Keywords miR-200a; angiogenesis; HUVECs; thrombospondin-1; cell viability; cell migration. Abbreviations ASO, antisense oligonucleotide; ECs, endothelial cells; EGFP, enhanced green fluorescence protein; HUVECs, human umbilical vein endothelial cells; miRNA, microRNA; THBS1, thrombospondin-1; UTR, untranslated region; VEGF, vascular endothe- lial growth factor. INTRODUCTION Angiogenesis is a process of growth of new blood vessels, which is a very complex process involving a number of molecu- lar and cellular regulators and keeping a finely tuned balance between stimulatory and inhibitory signals (1). Imbalance of this physiological process is involved in varieties of diseases such as cancer, diabetic retinopathy, thrombosis, and inflamma- tory disorders. MicroRNAs (miRNAs) are highly conserved small RNA molecules (22 nucleotides) which mostly regulate gene expression and participate in almost every cellular process (2). Recently, some labs have demonstrated that miRNAs play an important role in regulating the different aspects of angiogenic process (3). Evidence for the importance of microRNAs in the regulation of angiogenesis comes from observations that Dicer is required for embryonic angiogenesis and that knocking it out resulted in severely compromised embryos and yolk sacs (4). Silencing Dicer in adult endothelial cells (ECs) results in the downregulation of lef-7f and mir-27b which target antiangio- genic genes (5). Being consistent with this founding, changes in microRNA expression profiles in response to angiogenesis have been reported (5–7). This observation suggests that microRNA expression is generally necessary for angiogenesis. But the roles of these miRNAs in angiogenesis and endothelial function are totally different. MiR-126, miR-210, and miR-296 are consid- ered as proangiomiRs (8–10); while miR-221/222, miR-15, and miR-16 are believed to be anti-angiomiRs (4, 6, 11, 12). Endo- thelial miRNAs regulate the angiogenic response to multiple growth factors by targeting angiogenic factors, receptors and signaling molecules. In addition to their specific role in ECs phenotype and angiogenesis, dysregulation of some miRNAs contribute to tumor angiogenesis, such as miR-221/222, miR- 296, miR-210, and the miR-17-92 cluster (4, 6, 9–11, 13). At present, some endothelial miRNAs had been identified, but the role of miRNAs in angiogenesis is still little known. Here, we report that blocking miR-200a suppresses HUVECs viability and migration by targeting an angiogenesis inhibitor, thrombospondin-1 (THBS1). MATERIALS AND METHODS Cell Culture and Transfection Human umbilical vein endothelial cells (HUVECs) were cultured as previously described (14). For all experiments, Additional Supporting Information may be found in the online ver- sion of this article. *These authors contributed equally to this work. Received 9 February 2011; accepted 2 April 2011 Address correspondence to: Hua Tang, Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, No. 22 Qi-Xiang-Tai Road, Tianjin 300070, China. Tel: 186-22-23542603. Fax: 186-22-23542503. E-mail: htang2002@yahoo.com ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.486 IUBMB Life, 63(7): 553–559, July 2011