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

Abstract

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.

Supplemental material for this article may be found at http://mcb.asm.org/.

This work was supported by National Institutes of Health grant CA69184; Swiss National Science Foundation grant 3100A0-108207; Austrian Science Foundation grant S9408-B11; Cancer League Zürich, Commission of the European Communities, grant LSHC-CT-2005-518178 (M.D.); Swiss National Science Foundation grant 3100A0-101964 (A.W.B.); Netherlands Organization for Scientific Research (NWO) Venigrant (T.K.); and EMBO long-term fellowships ALTF 1104-2007 (D.M.L.P.) and ALTF 52-2007 (T.K.).

We thank Young Kwon Hong for the PROX1 3′ UTR plasmid (YH1551); Salvatore Oliviero, Jay W. Shin, and Ahmad Salameh for sharing the PROX1 lentivirus knockdown microarray data set; Patrick Pedrioli for bioinformatic assistance and critical reading of the manuscript; and Jana Zielinski, Cornelius Fischer, and Jeannette Scholl for expert technical assistance. We also thank the Tübingen 2000 Screen Consortium for identifying the plcg1^t26480 allele.

D.M.L.P. and T.K. designed and performed research experiments, analyzed the data, and wrote the manuscript. V.D., G.J., G.V.D.H., and R.E.K. designed and performed research experiments and analyzed the data. D.M., J.W.S., S.L., and P.C. performed research experiments and analyzed the data. M.D., A.W.B., and S.S.-M. designed research experiments, analyzed the data, and wrote the manuscript.