Abstract 18783: Regulation and Function of the Laminar Flow-induced Non-coding Rnas in Endothelial Cells
Recent analyses showed that only 2% of the human transcriptome represents protein coding sequences whereas a large fraction of non-coding RNA (ncRNA) rather exerts their function directly as RNA molecules. NcRNA can be classified into subgroups by length, classifying long non-coding RNA (lncRNAs) as transcripts longer than 200 nucleotides.
Based on the hypothesis that lncRNAs contribute to endothelial cell (EC) function, we assessed lncRNA expression in EC that are exposed to laminar flow (20 dynes/cm2) or static conditions by next generation deep sequencing. We chose 5 candidate lncRNAs and confirmed their regulation by shear stress by RT-qPCR analyses (lncflow1: 1.31±0.38; lncflow2: 3.35±1.51; lncflow3: 1.69±0.57; lncflow4: 12.69±1.57 and lncflow5: 0.16±0.06 fold change). Lentiviral over-expression of the flow induced transcription factor KLF2 also regulates the expression of lncflow2 (2.76±0.61 fold) and lncflow4 (3.41±0.16 fold, p<0.05).
Since lncflow2 was regulated by shear stress and KLF2 over-expression, we further explored its molecular and biological function. Transfection with LNA-GapmeRs, which reduced lncflow2 expression by 81.4±2.1% (p<0.05), induced spheroid sprouting of ECs (213±45% increase, p<0.05). Subcellular analyses indicated that lncflow2 is preferentially localized in the nucleus. Furthermore, lncflow2 was bound to the repressive chromatin mark H3K27me3 as shown by RNA immunoprecipitation suggesting that lncflow2 might epigenetically control gene expression. As some lncRNAs locally regulate gene expression, we analyzed the expression of neighbouring genes of lncflow2 by RT-qPCR. Inhibition of lncflow2 induced the expression of the metalloprotease AMZ2 (128±6%, p<0.05). Inhibition of AMZ2 by siRNA transfection reduces spheroid sprouting of ECs (60±19% compared to siRNA control) suggesting that lncflow2 regulates the expression of AMZ2 in cis. Indeed, siRNA mediated suppression of AMZ2 partially reversed the pro-angiogenic response observed after lncflow2 inhibition.
In summary, our data demonstrate that shear stress and KLF2 over-expression regulate the expression of lncRNAs in ECs. Inhibition of lncflow2 enhanced spheroid sprouting and induced the expression of the metalloprotease AMZ2.
Author Disclosures: C.T. Hartung: None. K.M. Michalik: None. X. You: None. W. Chen: None. A.M. Zeiher: None. R.A. Boon: None. S. Dimmeler: None.
- © 2014 by American Heart Association, Inc.