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Deep sequencing reveals predominant expression of miR‐21 amongst the small non‐coding RNAs in retinal microvascular endothelial cells
Author(s) -
GuduricFuchs Jasenka,
O'Connor Anna,
Cullen Angela,
Harwood Laura,
Medina Reinhold J.,
O'Neill Christina L.,
Stitt Alan W.,
Curtis Tim M.,
Simpson David A.
Publication year - 2012
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.24084
Subject(s) - mirbase , microrna , biology , small rna , deep sequencing , angiogenesis , gene knockdown , rna , genetics , non coding rna , microbiology and biotechnology , computational biology , gene , genome
The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non‐coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well‐characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR‐2284/2285 family were detected. Several ∼30 nucleotide sno‐miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR‐126 and miR‐378, but the most highly expressed was miR‐21, comprising more than one‐third of all mapped reads. Inhibition of miR‐21 with an LNA inhibitor significantly reduced proliferation, migration, and tube‐forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR‐21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature. J. Cell. Biochem. 113: 2098–2111, 2012. © 2012 Wiley Periodicals, Inc.