Abstract 15235: Characterization of Hypoxia-induced Long Non-coding RNAs in Endothelial Cells
RNA deep sequencing revealed that the majority of the genome is transcribed, however, only 2% of all transcripts encode for proteins. The remaining transcripts are referred to as non-coding RNAs and can be divided into small non-coding RNAs (<200nt) and long non-coding RNAs (lncRNAs; >200nt). Recent studies suggest that lncRNAs can regulate gene expression at various stages, e.g. by acting as scaffolds, by modulating splicing, or by chromatin modification. Regarding endothelial cells (EC), the response to hypoxia and the regulation of angiogenic activity are key events in the context of several diseases, however, the involvement of regulatory lncRNAs and their molecular function is still elusive.
To systematically determine the regulation of lncRNAs by hypoxia, we performed RNA-seq in HUVEC after exposure to hypoxia (24h; 0.2% O2) and identified 71 lncRNAs to be significantly up- (p≤0.01) and 78 lncRNAs to be down-regulated in comparison to normoxic conditions. qRT-PCR was used to validate the hypoxia-dependent regulation of 5 selected lncRNAs (Hyp_up_1 to Hyp_up_5). Since hypoxia controls the angiogenic function of EC, we next investigated whether our candidate lncRNAs affect endothelial cell sprouting as an in vitro measurement for angiogenic response. Silencing was achieved by GapmeRs (Hyp_up_1, 3) or siRNAs (Hyp_up_2, 5). Strikingly, only silencing of Hyp_up_1 significantly reduced sprouting under basal conditions and VEGF stimulation. Therefore, we selected Hyp_up_1 for further functional characterization: First, nuclear cytoplasmic fractionation revealed its predominant nuclear localization. Second, RNA immunoprecipitation (RIP) showed that Hyp_up_1 is mainly associated with repressive chromatin marks (3meH3K27) indicating that it may be involved in epigenetic gene regulation. Third, GO term analysis based on exon array data revealed a reduced expression of angiogenesis related genes upon Hyp_up_1 silencing. Finally, using antisense affinity purification and mass spectrometry, we identified several Hyp_up_1 binding proteins and confirmed their interaction by RIP. Hyp_up_1 bound proteins included PTBP1/U2AF65, two splicing regulators, as well as LOXL2, a transcription factor known to mediate angiogenic functions in EC.
Author Disclosures: P. Neumann: None. N. Jaé: None. D. John: None. S. Uchida: None. M. Krüger: None. S. Dimmeler: Consultant/Advisory Board; Modest; Miragen. Research Grant; Significant; ERC, DFG.
- © 2015 by American Heart Association, Inc.