Long noncoding RNA ANRIL regulates endothelial cell activities associated with coronary artery disease by up-regulating CLIP1, EZR, and LYVE1 genes

Abstract

Edited by Ronald C. Wek Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genomewide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development. Coronary artery disease (CAD) 4 is the most common cardiovascular disease and the leading cause of death worldwide (1). It occurs as a chronic inflammatory response to endothelial injuries in coronary arteries. Then the coronary arteries develop an atherosclerotic plaque inside the intima (referred to as atherosclerosis), leading to hardening and narrowing of the arteries and blocking of blood flow to the heart. Typical CAD complications include stable angina, unstable angina, myocardial infarction (MI), arrhythmias, heart failure, and sudden death (2). For the development of atherosclerosis and CAD, monocyte adhesion to the endothelium and transendothelial migration of monocytes (TEM) into the intima have been established as two of the most important cellular processes by multiple studies (1, 2). An increased serum concentration of oxidized low-density lipoproteins (ox-LDLs) or other inflammatory molecules such as TNF␣ recruits monocytes to the region where ROS-induced endothelial dysfunction occurs (1-3). This leads to monocyte adhesion to the endothelium, followed by transmigration of monocytes across the endothelium into the intima area (1-3). The monocytes are then transformed into lipid-engorged macrophages and foam cells, leading to the formation of plaques (1-3).

Relevance of Multi-Omics Studies in Cardiovascular Diseases

“…This process is mediated through the expression of multiple genes regulated in both, cis and trans (69, 70). Moreover, a recent study showed that ANRIL (DQ485454) is involved in endothelial cells functions important to the development of CAD including monocyte adhesion to endothelial cells, trans-endothelial monocyte migration, and endothelial cell migration (71).…”

Conceptualization of Heterogeneity of Chronic Diseases and Atherosclerosis as a Pathway to Precision Medicine: Endophenotype, Endotype, and Residual Cardiovascular Risk

“…The following genes involved in the regulation of various pathways of inflammation, according to various GWAS, are significantly associated with atherosclerotic cardiovascular diseases (CVD): CXCL12, MCP-1, TLRs, SH2B3, HLA, IL-6R, IL-5, PECAM1, and others [70]. In studies based on the proteomics approach, the crucial role of inflammation was also shown by the example of the activation of the TNF-α pathway in the vascular wall, as well as the migration of monocytes into the subendothelial compartment (ANRIL) [71,72].…”

The Long Non-Coding RNA Landscape of Atherosclerotic Plaques

“…Interestingly, the DQ485454 transcript was most abundant in EC lines and decreased in the coronary artery of CAD patients. Gain-and loss-of-function experiments revealed that ANRIL DQ485454 affects monocyte adhesion to ECs and transmigration across ECs along with EC migration, probably via regulation of the expression of CLIP1 , EZR , and LYVE1 [51]. Guo et al [52] have shown that human coronary artery ECs (HCAECs) viability, survival and apoptosis, and the production of proteins specific for the epithelial–mesenchymal transition along with the release of inflammatory factors and vascular-protective factors might be regulated by the ANRIL /miR-181b/NF-κB signalling pathway [52].…”