Premium
Deep Sequencing and Bioinformatics Analysis of Endothelial MicroRNA under Hypoxia Stress
Author(s) -
Chen Zhen,
Sun Wei,
Padmanabhan Chellappan,
Wang WeiChi,
Jin Hailing,
Huang HsienDa,
Chien Shu,
Shyy John YJ
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.784.10
Subject(s) - microrna , biology , in silico , gene , rna , deep sequencing , hypoxia (environmental) , gene expression , transcription factor , small rna , messenger rna , microbiology and biotechnology , genetics , genome , chemistry , organic chemistry , oxygen
We hypothesized that MicroRNAs (miRs) play an important role in endothelial responses to hypoxia. In order to identify miRNAs and to assess their expression patterns, small RNAs extracted from cultured human umbilical vein endothelial cells under normoxia or hypoxia (2% O 2 ) were ligated with distinct RNA adaptors and underwent RT‐PCR to establish the cDNA library, which was then subjected to sequencing‐by‐synthesis deep sequencing, generating 10 6 small RNA sequences. Among 157 miRs identified, 41 were up‐ and 16 down‐regulated. Of interest, two highly conserved miR families were robustly induced by hypoxia, as validated by Northern blot. With the use of position weight matrix from TRANSFAC database and a prediction algorithm combining miRanda‐RNAhybrid‐TargetScan, both the transcription factors and the mRNA targets of these miRs were predicted. Hypoxia‐inducible factor 1α may target these miRs through hypoxia‐responsive elements located in the putative promoter region of these miRNA genes. As a consequence, these hypoxia‐induced miRs lead to the suppression of a panel of putative target genes, which were further verified by gain‐ or loss‐of‐function experiments using pre‐miRs or locked nucleic acids. By utilizing a strategy combining in silico and molecular and cellular experiments, we demonstrated that HIF1α can regulate endothelial miRs which in turn modulate downstream gene expression.