miR-31 Functions as a Negative Regulator of Lymphatic Vascular Lineage-Specific Differentiation In Vitro and Vascular Development In Vivo

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zürich, Wolfgang-Pauli-Str. 10, HCI H303, CH-8093 Zürich, Switzerland.
Molecular and Cellular Biology (Impact Factor: 4.78). 07/2010; 30(14):3620-34. DOI: 10.1128/MCB.00185-10
Source: PubMed


The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses, and promotes cancer
metastasis. To address the role microRNAs (miRNAs) play in the development and function of the lymphatic vascular system,
we defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs)
and identified four BVEC signature and two LEC signature miRNAs. Their vascular lineage-specific expression patterns were
confirmed in vivo by quantitative real-time PCR and in situ hybridization. Functional characterization of the BVEC signature miRNA miR-31 identified a novel BVEC-specific posttranscriptional
regulatory mechanism that inhibits the expression of lymphatic lineage-specific transcripts in vitro. We demonstrate that suppression of lymphatic differentiation is partially mediated via direct repression of PROX1, a transcription
factor that functions as a master regulator of lymphatic lineage-specific differentiation. Finally, in vivo studies of Xenopus and zebrafish demonstrated that gain of miR-31 function impaired venous sprouting and lymphatic vascular development, thus
highlighting the importance of miR-31 as a negative regulator of lymphatic development. Collectively, our findings identify
miR-31 is a potent regulator of vascular lineage-specific differentiation and development in vertebrates.

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Available from: André W Brändli, May 05, 2014
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    • "Targeted functions of hsa-miR-31 include angiogenesis, where its upregulation increases blood, but not lymphatic vessels sprouting [48], and acute inflammation [49]. We found that hsa-miR-31 is mostly up-regulated in peritumoral inflammatory cells, compared to cancer and normal cells. "
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    ABSTRACT: MicroRNAs are being exploited for diagnosis, prognosis and monitoring of cancer and other diseases. Their high tissue specificity and critical role in oncogenesis provide new biomarkers for the diagnosis and classification of cancer as well as predicting patients' outcomes. MicroRNAs signatures have been identified for many human tumors, including colorectal cancer (CRC). In most cases, metastatic disease is difficult to predict and to prevent with adequate therapies. The aim of our study was to identify a microRNA signature for metastatic CRC that could predict and differentiate metastatic target organ localization. Normal and cancer tissues of three different groups of CRC patients were analyzed. RNA microarray and TaqMan Array analysis were performed on 66 Italian patients with or without lymph nodes and/or liver recurrences. Data obtained with the two assays were analyzed separately and then intersected to identify a primary CRC metastatic signature. Five differentially expressed microRNAs (hsa-miR-21, -103, -93, -31 and -566) were validated by qRT-PCR on a second group of 16 American metastatic patients. In situ hybridization was performed on the 16 American patients as well as on three distinct commercial tissues microarray (TMA) containing normal adjacent colon, the primary adenocarcinoma, normal and metastatic lymph nodes and liver. Hsa-miRNA-21, -93, and -103 upregulation together with hsa-miR-566 downregulation defined the CRC metastatic signature, while in situ hybridization data identified a lymphonodal invasion profile. We provided the first microRNAs signature that could discriminate between colorectal recurrences to lymph nodes and liver and between colorectal liver metastasis and primary hepatic tumor.
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    • "We previously established the Xenopus laevis tadpole as a genetic model for lymphangiogenesis research, phenocopying deficiencies of known mammalian lymphatic genes (Ny et al., 2005). We and others further applied the tadpole model to investigate molecular regulation of lymphatic vascular development, including its use in chemical library screens to identify anti-lymph/angiogenesis compounds (Kälin et al., 2009; Marino et al., 2011; Ny et al., 2008; Leslie Pedrioli et al., 2010). In these studies, visualization of the blood-and lymphatic vasculature depended on staining by in situ hybridization (ISH). "
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    ABSTRACT: The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.
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    • "One of the CB-EPC-dominant microRNAs is miR-31, a pro-angiogenic miRNA that enhances endothelial cell migration [18,37]. MiR-31 has recently been documented as a signature BEC miRNA that negatively regulates lymphatic endothelial cell identity and lymphatic vascular development by targeting Prox1, a transcription factor that functions as a master regulator of lymphatic lineage-specific differentiation [45]. In the present study, we further showed that miR-31 is a dominant miRNA in CB-EPCs, and its overexpression is crucial for EPCs to possess superior angiogenic ability (Figure 4). "
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