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

• Authors: Lauren G. Douma, University of Florida; Kristen Solocinski, University of Florida; Sarah H. Masten, University of Florida; Dominique H. Barral, University of Florida; Sarah J. Barilovits, University of Florida; Lauren Jeffers, Emory University; Kareme D. Alder, Yale University; Ravi Patel, University of Florida; Charles S. Wingo, University of Florida; Kevin D. Brown, University of Florida; Brian D. Cain, University of Florida; Michelle L. Gumz, University of Florida
• Journal Title: Front Physiol
• Volume: Volume 11
• Publisher: Frontiers | 2020-03-13, Pages 209-None
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.3389/fphys.2020.00209
• Subjects: Health Sciences, Medicine and Surgery; Biology, Genetics
• Keywords: kidney; long non-coding RNA; proximal tubule cells; CRISPR; circadian rhythm

Abstract

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, EDN1. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the EDN1 gene, called EDN1-AS. Because EDN1-AS represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of EDN1-AS expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted EDN1-AS 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 EDN1-AS, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, n = 7, P < 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 (n = 4, P < 0.01) and the KO2 clone exhibited a two-fold increase (n = 4, P < 0.01). These results support a role for EDN1-AS as a novel regulatory mechanism of ET-1 expression and cellular proliferation.