AUTHOR=Douma Lauren G. , Solocinski Kristen , Masten Sarah H. , Barral Dominique H. , Barilovits Sarah J. , Jeffers Lauren A. , Alder Kareme D. , Patel Ravi , Wingo Charles S. , Brown Kevin D. , Cain Brian D. , Gumz Michelle L. 

TITLE=EDN1-AS, A Novel Long Non-coding RNA Regulating Endothelin-1 in Human Proximal Tubule Cells

JOURNAL=Frontiers in Physiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2020.00209

DOI=10.3389/fphys.2020.00209

ISSN=1664-042X

ABSTRACT=<p>Endothelin-1 (ET-1) is a peptide hormone that functions as a vasoconstrictor in the vasculature, whereas in the collecting duct of the kidney it exerts blood pressure-lowering effects via natriuretic actions. Aberrant ET-1 signaling is associated with several pathological states including hypertension and chronic kidney disease. ET-1 expression is regulated largely through transcriptional control of the gene that encodes ET-1, <italic>EDN1</italic>. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the <italic>EDN1</italic> gene, called <italic>EDN1-AS</italic>. Because <italic>EDN1-AS</italic> represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of <italic>EDN1-AS</italic> expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted <italic>EDN1-AS</italic> transcription start site was identified using the ENCODE database and the UCSC genome browser. Two homozygous deletion clones of the element were generated using CRISPR/Cas9. This deletion resulted in a significant increase in the expression of <italic>EDN1-AS</italic>, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, <italic>n</italic> = 7, <italic>P</italic> &lt; 0.05). Phenotypic characterization of these CRISPR clones revealed a difference in cell growth rates. Using a standard growth assay, we determined that the KO1 clone exhibited a three-fold increase in growth over 8 days compared to control cells (<italic>n</italic> = 4, <italic>P</italic> &lt; 0.01) and the KO2 clone exhibited a two-fold increase (<italic>n</italic> = 4, <italic>P</italic> &lt; 0.01). These results support a role for <italic>EDN1-AS</italic> as a novel regulatory mechanism of ET-1 expression and cellular proliferation.</p>