miR-200b Targets Ets-1 and Is Down-regulated by Hypoxia to Induce Angiogenic Response of Endothelial Cells

Abstract

Micro-RNAs (miRs)2 are small non-coding RNAs consisting of ϳ22 nucleotide base pairs. These small nucleic acids could regulate gene expression by binding to the 3Ј untranslated region (3Ј UTR) of mRNA, resulting in either translational repression or transcript degradation. Around 30% of the genes in the whole genome are subjected to regulation by miRs (1). Because of the short sequence requirement for binding the target 3Ј UTR, a single miR could interact with a wide range of target transcripts, which could substantially alter gene expression and dictate cell fate such as proliferation, apoptosis, and cell migration.Angiogenesis, the formation of vessels from the existing vascular structure, is a crucial biological response in wound healing, menstrual cycle, tumor aggression, and diabetic retinopathy. Endothelial sprouting, which requires extracellular matrix remodeling and endothelial cell migration, is the prerequisite process of angiogenic response. Such biological response relies on highly coordinated gene expression in a temporal and spatial manner. Increasing evidence revealed that miRs play a pivotal role in the angiogenic process. Our group and others reported that dicer, a ribonuclease III catalyzing miR maturation, is involved in the angiogenic process in human endothelial cells (2-4). Endothelial-specific dicer knockout mice exhibited an impaired angiogenic response (5). Certain miRs such as miR-126 and miR-296 have been shown to exert pro-angiogenic effects, whereas other reports indicated that miR-130a, -221, and -222 inhibited angiogenesis (6). Recently, the miR-200 family has been shown to arrest cell migration and modulate epithelial-mesenchymal transition in a wide range of epithelial cancer cells (7-12). We hypothesized that miR-200b, one member in miR-200 family, regulates endothelial cell migration and angiogenic responses. In this study, we present the first evidence demonstrating that miR200b is hypoxia-inducible and down-regulates v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), a novel direct target of miR-200b, subsequently promoting angiogenic response of HMEC. EXPERIMENTAL PROCEDURESCells, Cell Culture, and Hypoxic Treatment-Human dermal microvascular endothelial cells (HMECs) were cultured in a humidified chamber (37°C, 20% O 2 and 5% CO 2 ) in MCDB-131 medium supplemented with 10% FBS, 10 mM Lglutamine, and 100 IU/ml of penicillin, 0.1 mg/ml of streptomycin (Invitrogen), as described previously (3). Primary adult human dermal microvascular endothelial cells were cultured at 37°C (20% O 2 and 5% CO 2 ) in EBM-2 medium (Lonza) supplemented with EGM-2MV single quotes (Lonza) as described by the manufacturer. HEK-293 cells were grown at standard cell culture conditions (37°C, 20% O 2 and 5% CO 2 ) with DMEM supplemented with 10% FBS and 100 IU/ml of penicillin, 0.1 mg/ml of streptomycin. For hypoxic treatment, cells were seeded on a 35-mm dish at 0.5 ϫ 10 6 cells/plate 1 * This work was supported, in whole or in part, by National Institutes of Health Grants GM077185 and GM069...

Regulation of Vascular Endothelial Growth Factor Signaling by miR-200b

“…As stated earlier, miR-200b targets Ets-1 and inhibits angiogenic activity of HMECs (Chan et al, 2011). Another paper reported that miR-200b targets murine Flt-1 (Roybal et al, 2011).…”

“…In vivo, knockdown of miR-126 in mice resulted in leaky vessels, hemorrhaging, and partial embryonic lethality, suggesting that miR-126 functions as a positive regulator of angiogenesis. In the case of antiangiogenic miRNAs, miR-200b targets v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), thereby inhibiting the angiogenic response of human microvascular endothelial cells (HMECs) (Chan et al, 2011). When transfected with miR-200b, the ability of HMECs to migrate and to form tubes on Matrigel was inhibited.…”

miRNA-145 Targets v-ets Erythroblastosis Virus E26 Oncogene Homolog 1 to Suppress the Invasion, Metastasis, and Angiogenesis of Gastric Cancer Cells

“…For example, miR-125b is downregulated in invasive breast cancers, and overexpression of miR-125b inhibits the tumorigenesis by targeting Ets1 in breast cancer cells (38). Hypoxia-sensitive miR-200b is involved in induction of angiogenesis via directly targeting Ets1 in endothelial cells (39). Ectopic expression of miR193b dramatically suppresses the proliferation, migration, and invasion of hepatocellular carcinoma cells by targeting Ets1 and cyclin D1 (40).…”

miRNAs and lncRNAs in vascular injury and remodeling

“…These miRNAs include miR-21, miR-24, miR-34a, miR-92a, miR-126, miR-200b, miR-210, and miR-221/222 cluster ( Figure 2). Many of these miRNAs inhibit angiogenesis by suppressing proliferation and migration of endothelial cells, such as miR-21 by targeting proliferators-activated receptor-alpha (PPAR-α) and RhoB [27]; miR-200b by down-regulating Ets-1, VEGF, VEGFR-2 and GATA2 [28]; miR-92a by suppressing integrin subunit a5 [29]; and miR-221/222 by negatively regulating the expression of c-kit [30]. Moreover, in addition to suppressing angiogenesis by regulating GATA2, miR-24 also induces apoptosis by targeting p21-activating kinase 4 (PAK4) [31].…”