Regulation of CCL2 expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA

Abstract

Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)− regulated messenger RNA (mRNA)−lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2, which is transcribed divergently to the gene, CCL2, encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA−lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2, which may contribute to atherogenesis in humans.