Genomic Landscapes of Noncoding RNAs Regulating VEGFA and VEGFC Expression in Endothelial Cells.

Isidore Mushimiyimana,Minna U Kaikkonen,Pierre R Moreau,Nicholas L Downes,Vanesa Tomas Bosch,Kiley Hartigan,Seppo Ylä-Herttuala,Henri Niskanen,Nihay Laham-Karam

doi:10.1128/mcb.00594-20

Abstract

ABSTRACTVascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. Although VEGFs have been promising therapeutic targets for various cardiovascular diseases, their regulatory landscape in endothelial cells remains elusive. Several studies have highlighted the involvement of noncoding RNAs (ncRNAs) in the modulation of VEGF expression. In this study, we investigated the role of two classes of ncRNAs, long ncRNAs (lncRNAs) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC. By integrating genome-wide global run-on sequencing (GRO-Seq) and chromosome conformation capture (Hi-C) data, we identified putative lncRNAs and eRNAs associated with VEGFA and VEGFC genes in endothelial cells. A subset of the identified putative enhancers demonstrated regulatory activity in a reporter assay. Importantly, we demonstrate that deletion of enhancers and lncRNAs by CRISPR/Cas9 promoted significant changes in VEGFA and VEGFC expression. Transcriptome sequencing (RNA-Seq) data from lncRNA deletions showed downstream factors implicated in VEGFA- and VEGFC-linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells.

Highlights

Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis
The vascular endothelial growth factor (VEGF) family is composed of 5 members in mammals: VEGF-A, -B, -C, and -D and placental growth factor (PlGF) [1,2,3,4,5], which exert their functions through three tyrosine kinase receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR/FLK-1), and VEGFR-3 (Flt4) [6,7,8]
By using next-generation sequencing data from endothelial cells, we identified novel enhancer RNAs (eRNAs) and long noncoding RNAs (ncRNAs) (lncRNAs) that interact with VEGFA and VEGFC promoters

Summary

Introduction

Vascular endothelial growth factors (VEGFs) are best known as key regulators of angiogenesis and lymphangiogenesis. We investigated the role of two classes of ncRNAs, long ncRNAs (lncRNAs) and enhancer RNAs (eRNAs), in the transcriptional regulation of VEGFA and VEGFC. Transcriptome sequencing (RNA-Seq) data from lncRNA deletions showed downstream factors implicated in VEGFA- and VEGFC-linked pathways, such as angiogenesis and lymphangiogenesis, suggesting functional roles for these lncRNAs. Our study uncovers novel lncRNAs and eRNAs regulating VEGFA and VEGFC that can be targeted to modulate the expression of these important molecules in endothelial cells. Other external signals, such as growth factors and interleukins (ILs), can induce VEGF expression [14] Due to their crucial role in regulating endothelial cell behavior, VEGFs are therapeutic targets for many diseases, such as cardiovascular diseases [15] and cancer [16]. We have come to appreciate the complexity of transcriptional regulation and the role of noncoding RNAs (ncRNAs) in this process [17]

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