Long non-coding RNA CDKN2B-AS1 contributes to atherosclerotic plaque formation by forming RNA-DNA triplex in the CDKN2B promoter

EBioMedicine. 2020 May:55:102694. doi: 10.1016/j.ebiom.2020.102694. Epub 2020 Apr 24.

Abstract

Background: Atherosclerosis involves a slow process of plaque formation on the walls of arteries, and comprises a leading cause of cardiovascular disease. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of lncRNA 'cyclin-dependent kinase inhibitor 2B antisense noncoding RNA' (CDKN2B-AS1) and CDKN2B in the progression of atherosclerosis.

Methods: Initially, we quantified the expression of CDKN2B-AS1 in atherosclerotic plaque tissues and, in THP-1 macrophage-derived, and human primary macrophage (HPM)-derived foam cells. Next, we established a mouse model of atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice, where lipid uptake, lipid accumulation, and macrophage reverse cholesterol transport (mRCT) were assessed, in order to explore the contributory role of CDKN2B-AS1 to the progression of atherosclerosis. RIP and ChIP assays were used to identify interactions between CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2), and CDKN2B. Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment.

Findings: CDKN2B-AS1 showed high expression levels in atherosclerosis, whereas CDKN2B showed low expression levels. CDKN2B-AS1 accelerated lipid uptake and intracellular lipid accumulation whilst attenuating mRCT in THP-1 macrophage-derived foam cells, HPM-derived foam cells, and in the mouse model. EZH2 and CTCF were found to bind to the CDKN2B promoter region. An RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in the CDKN2B promoter region and consequently inhibit CDKN2B transcription by accelerating histone methylation.

Interpretation: The results demonstrated that CDKN2B-AS1 promotes atherosclerotic plaque formation and inhibits mRCT in atherosclerosis by regulating CDKN2B promoter, and thereby could be a potential therapeutic target for atherosclerosis.

Keywords: Atherosclerosis; Cyclin-dependent kinase inhibitor 2B; Epigenetics; Long non-coding RNA CDKN2B-AS1; Macrophage reverse cholesterol transport; RNA-DNA triplex.

MeSH terms

  • Animals
  • Apolipoproteins E / deficiency
  • Apolipoproteins E / genetics
  • Atherosclerosis / genetics*
  • Atherosclerosis / metabolism
  • Atherosclerosis / pathology
  • Base Sequence
  • Biological Transport
  • CCCTC-Binding Factor / genetics
  • CCCTC-Binding Factor / metabolism
  • Cholesterol / metabolism*
  • Cyclin-Dependent Kinase Inhibitor p15 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p15 / metabolism
  • DNA / genetics
  • DNA / metabolism
  • Disease Models, Animal
  • Enhancer of Zeste Homolog 2 Protein / genetics
  • Enhancer of Zeste Homolog 2 Protein / metabolism
  • Epigenesis, Genetic
  • Foam Cells / metabolism
  • Foam Cells / pathology
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Lipid Metabolism / genetics
  • Methylation
  • Mice
  • Mice, Knockout
  • Plaque, Atherosclerotic / genetics*
  • Plaque, Atherosclerotic / metabolism
  • Plaque, Atherosclerotic / pathology
  • Primary Cell Culture
  • Promoter Regions, Genetic*
  • RNA, Long Noncoding / genetics*
  • RNA, Long Noncoding / metabolism
  • THP-1 Cells

Substances

  • Apoe protein, mouse
  • Apolipoproteins E
  • CCCTC-Binding Factor
  • CDKN2B antisense RNA, human
  • CDKN2B protein, human
  • CTCF protein, human
  • Cyclin-Dependent Kinase Inhibitor p15
  • Histones
  • RNA, Long Noncoding
  • DNA
  • Cholesterol
  • EZH2 protein, human
  • Enhancer of Zeste Homolog 2 Protein