Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs

Abstract

In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% % O 2 or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40% % of all miRNAs. A complex repertoire of isomiRs was found, displaying also 59 variations, potentially affecting target recognition. Highstringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR-210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxiainducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia.

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

“…by guest, on www.jlr.org Previously reported deep-sequencing results identifi ed 427 known and novel miRNAs expressed in HUVECs ( 41 ). Our study identifi ed 369 of these 427 miRNAs.…”

A Regulatory Role for MicroRNA 33* in Controlling Lipid Metabolism Gene Expression

“…Our present study, however, suggests that the accumulation of miR33a* and miR-33b* is tissue dependent, a finding that both challenges this hypothesis and extends recent reports of miRNA tissue-dependent stability. Indeed, while this article was in preparation, Voellenkle et al reported a clear bias toward miR-33a* accumulation in endothelial cells using deep sequencing techniques (38). Additionally, it was reported that miR-33a* was not only expressed in monocyte-derived macrophages (MDMs), but also downregulated under M2-polarizing conditions (39).…”

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

“…MicroRNAs are now established therapeutically viable targets in the regulation of vascular inflammation and senescence (e.g., miR-146a, miR-217, miR-34a, miR-126, miR-21, miR-210, miR-181b) (121,151,169,170), tumor angiogenesis (miR-19b-1) (191), and in vascular diseases such as hypertension (miR-125a/b-5p) (83) and atherosclerosis (miR-92a, miR-27, miR-10a) (23, 46,47). In view of the established roles for TM in the regulation of inflammatory and thrombotic processes within the vascular wall, in conjunction with its indirect regulation by miR-92a via KLF2 (46,187), one can anticipate future studies detailing approaches to modulating TM levels in vivo using miRNA-targeting strategies.…”