Abstract 15654: The Hypoxia-induced Long Non-coding RNA Hyp_up_3 Regulates Alternative Splicing and Controls Angiogenesis of Endothelial Cells
RNA deep sequencing revealed that the human genome is pervasively transcribed, however, the vast majority are non-coding transcripts and belong to the class of long non-coding RNAs (lncRNAs). LncRNAs are known to have various functions like recruitment of chromatin modifiers, miRNA-sponging or modulation of mRNA splicing. In endothelial cells (ECs), the hypoxic response and the regulation of angiogenic activity are key events in the context of several diseases. However, the regulatory role and the molecular function of lncRNAs during these events remains obscure.
To systematically investigate the influence of hypoxia on lncRNAs expression, we applied RNA deep sequencing on HUVECs, subjected to hypoxia (24h; 0.2% O2). Data analysis resulted in the identification of 71 lncRNAs to be significantly up- (p≤0.01) and 78 lncRNAs to be down-regulated in comparison to normoxic conditions. We used qRT-PCR to validate the hypoxia dependent regulation of 5 selected lncRNAs (Hyp_up_1-5). Since hypoxia controls the angiogenic function of ECs, we next investigated whether these lncRNAs affect endothelial cell sprouting as an in vitro measurement for angiogenic response. By using LNA GapmeRs we could efficiently reduce Hyp_up_3 expression (30% ±5.63; p≤0.05) and significantly inhibit endothelial cell sprouting (60% ±7.06; p≤0.05). Also Hyp_up_3 silencing (53.38% ±5.54; p≤0.05) resulted in increased G0/1-phase arrest. Therefore, we focused on Hyp_up_3 for a more detailed characterization. Subcellular fractionation revealed a predominant nuclear localization. By using antisense affinity purification and mass spectrometry, we identified several Hyp_up_3 bound splicing regulators, including hnRNP L and DAZAP1. Both proteins act by binding to cis-regulatory sequences within pre-mRNAs to control alternative splicing. This is in line with microarray data from Hyp_up_3- silenced ECs showing more than 400 alternatively spliced genes compared to controls. GO-term analysis of these genes displayed their association with hypoxic response and angiogenesis. In conclusion, our data demonstrates that the hypoxia-regulated lncRNA Hyp_up_3 controls sprouting angiogenesis and endothelial cell cycle putatively through by modulating alternative splicing.
Author Disclosures: P. Neumann: None. Y. Fouani: None. N. Jaé: None. A. Knau: None. D. John: None. S. Uchida: None. J. Heidler: None. I. Wittig: None. S. Dimmeler: None.
- © 2016 by American Heart Association, Inc.