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PMID: 22282338 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

RNA (New York, N.Y.) ·Vol. 18 ·No. 3 ·2012-03-00 ·Pages 472-84

Voellenkle C, Rooij Jv, Guffanti A, Brini E, Fasanaro P, Isaia E, Croft L, David M, Capogrossi MC, Moles A, Felsani A, Martelli F

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% O₂ 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 5' variations, potentially affecting target recognition. High-stringency 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 hypoxia-inducible 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.

MeSH Terms
Carboxypeptidases/metabolism Cell Hypoxia Cell Proliferation Endothelial Cells/metabolism Gene Expression Profiling Gene Expression Regulation Gene Library HEK293 Cells High-Throughput Nucleotide Sequencing Humans MicroRNAs/analysis,chemistry,metabolism Molecular Sequence Annotation Nucleic Acid Conformation RNA, Double-Stranded Sequence Analysis, RNA Signal Transduction
Chemicals
MicroRNAs RNA, Double-Stranded Carboxypeptidases SCPEP1 protein, human
Authors & Affiliations
12 authors, click to expand affiliations / ORCID
Voellenkle Christine
Laboratorio di Cardiologia Molecolare, IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy.
Rooij Jeroen van
Guffanti Alessandro
Brini Elena
Fasanaro Pasquale
Isaia Eleonora
Croft Larry
David Matei
Capogrossi Maurizio C
Moles Anna
Felsani Armando
Martelli Fabio
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.027615.111
Published
2012-03-00
Epub
2012-00-26
Pages
472-84
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC3285935
Subset
IM
External Links
PubMed source DOI: 10.1261/rna.027615.111 PMC Full Text View journal details
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