Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans-Regulation of Gene Networks

Abstract

The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) antisense non-coding RNA in the INK4 locus (ANRIL). ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

The interplay of LncRNA ANRIL and miR‐181b on the inflammation‐relevant coronary artery disease through mediating NF‐κB signalling pathway

“…Zhou et al34 firstly disclosed that ANRIL and related inflammatory factors were up‐regulated within ECs under the stimulation of pathogens, which was mediated by up‐regulated TNF‐α that was caused by NF‐κB signalling. Also ANRIL was capable of binding to PRC‐associated protein (ie, Yin Yang 1) to induce or restrain genetic expressions (eg, IL6 and IL8) that were related with inflammatory responses 35, 36, 37. Thus, it was quite acceptable that ANRIL could act on NF‐κB signalling to induce inflammation, thereby generating CAD‐specific cell activities (Figures 3, 4, 5, 6, 7, 8).…”

The impact of genome‐wide association studies on the pathophysiology and therapy of cardiovascular disease

“…The chromosome 9p21 locus is a prominent example: whereas initial studies focused on the two genes situated at the locus, CDKN2A and CDKN2B, current studies rather point to radically new disease mechanisms. Indeed, it appears that the risk/non‐risk alleles at the 9p21 locus relate to different isoforms of ANRIL and subsequently to preferred synthesis of non‐circular/circular forms of this long non‐coding RNA, which affect via ribosomal function the expression of multiple genes leading ultimately to opposing effects on cell proliferation and apoptosis (Holdt et al , 2010, 2013). …”

Long Noncoding RNAs in Atherosclerosis

“…Katharina et al 51) found that MALAT1 silencing by siRNA or LNA GapmeRs induced a phenotype switch of the endothelial cells from a proliferation state to a promigratory state. This type of phenotype switch results in a block in vessel outgrowth cells and tissues such as SMCs, ECs, monocytederived macrophages, and RNA samples extracted from carotid and arterectomy 41) . Moreover, the severity of atherosclerosis with ANRIL expression has been described 39,42) .…”

“…Moreover, Holdt et al found that Alu motifs are essential for the pro-atherogenic functions of ANRIL. ANRIL regulates target genes through the Alu motifs located in ANRIL and the promoters of target-genes resulting in increased cell proliferation, cell adhesion, and decreased apoptosis, which all play critical roles in the process of atherosclerosis 41) .…”