Alaqeeli Maha, Mayaki Dominique, Hussain Sabah N A
Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Department of Critical Care, McGill University Health Centre, Montréal, QC, Canada.
Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, QC, Canada.
Front Physiol. 2021 Oct 21;12:729157. doi: 10.3389/fphys.2021.729157. eCollection 2021.
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. 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. 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. 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. 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.
长链非编码RNA（lncRNAs）是一类长度超过200个核苷酸的非编码RNA。它们最近已成为血管生成的重要调节因子。为了鉴定可能参与血管生成调节的新型lncRNAs，我们检测了暴露于缺氧环境24小时的人脐静脉内皮细胞（HUVECs）中84种lncRNAs的mRNA。其中之一，横纹肌肉瘤2相关转录本（RMST），在缺氧条件下显著上调。关于RMST在血管内皮细胞功能中的存在和作用知之甚少。本研究的主要目的是研究RMST在血管内皮细胞中的调控机制，并确定其在血管内皮细胞存活、增殖、迁移和分化中的作用。使用qPCR检测了HUVECs中10种RMST异构体的基础mRNA水平。然后分别检测了在缺氧24小时、共培养7天的分化过程以及暴露于四种不同血管生成因子后的水平。通过功能缺失方法（用单链反义LNA GapmeRs转染）定量了RMST在血管内皮细胞存活、迁移和分化中的功能作用。使用细胞计数和结晶紫试验检测血管内皮细胞存活情况。分别使用划痕伤口愈合试验和基质胶分化试验检测细胞迁移和分化情况。在HUVECs和人微血管内皮细胞（HMEC-1s）中检测到了五种RMST异构体（RMST-202、-203、-204、-206和-207）。其他类型的血管细胞，包括人主动脉瓣间质细胞和人主动脉平滑肌细胞，未显示出这种表达谱。在缺氧24小时以及HUVECs与人肺成纤维细胞共培养7天后，RMST显著上调。血管生成素-2（Ang-2）可显著下调RMST，但血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（FGF-2）或血管生成素-1（Ang-1）则无此作用。选择性敲低RMST表明，它可促进血管内皮细胞在血清剥夺条件下的存活。它也是VEGF和Ang-1诱导的血管内皮细胞存活和迁移所必需的，但对分化并非必需。我们得出结论，RMST在人血管内皮细胞中表达，并且这种表达在缺氧和分化为毛细血管样结构的过程中上调。我们还得出结论，RMST在血管内皮细胞存活和迁移中起重要作用。
