Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Maha Alaqeeli,Sabah N A Hussain,Dominique Mayaki

doi:10.3389/fphys.2021.729157

Abstract

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function.Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation.Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively.Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation.Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

Highlights

Angiogenesis is the formation of new blood vessels from pre-existing blood vessels
The short version, where transcripts have less than 200 nucleotides, is known as microRNAs, and their roles in angiogenesis have been extensively documented over the past several years. ncRNAs that have more than 200 nucleotides are known as long non-coding RNAs
Our study demonstrates for the first time that: 1) several rhabdomyosarcoma 2-associated transcript (RMST) isoforms are expressed in endothelial cells (ECs) and they are localized to the nucleus; 2) endothelial RMST expression is significantly upregulated during differentiation of ECs into capillary-like structures and in response to acute hypoxia; 3) exposure to Ang-2 but not vascular endothelial growth factor (VEGF), Ang-1, or FGF2 downregulates RMST expression in ECs; 4) RSMT plays a significant role in the pro-survival effects of VEGF and Ang-1; and 5) RMST promotes EC survival, migration, and proliferation

Summary

Introduction

Angiogenesis is the formation of new blood vessels from pre-existing blood vessels. It is of paramount physiological importance to normal embryonic vascular development, adult vascular regeneration and repair, and the vascular remodeling that is associated with diseases, such as atherosclerosis, pulmonary fibrosis, and tumor growth (Carmeliet, 2003). Epigenetic regulators of gene expression known as non-coding RNAs (ncRNAs) have emerged as important modulators of angiogenesis. Based on their length, ncRNAs can be characterized as either short or long. NcRNAs that have more than 200 nucleotides are known as long non-coding RNAs (lncRNAs). Several, including MALAT1, TUG1, MEG3, and LINC00657, have been shown to be abundantly expressed in ECs. Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides They have recently emerged as important regulators of angiogenesis. Little is known about the presence and roles of RMST in EC function

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