Long non-coding RNA CDKN2B-AS1 contributes to atherosclerotic plaque formation by forming RNA-DNA triplex in the CDKN2B promoter
Authors: Jie Tu
Minghui Ou
Shibo Zhao
Shichao Cui
Xia Li
Journal: EBioMedicine (2020)
Discipline: CLINICAL MEDICINE
Keywords: Atherosclerosis; Cyclin-dependent kinase inhibitor 2B; Epigenetics; Long non-coding RNA CDKN2B-AS1; Macrophage reverse cholesterol transport
Affiliations: Department of Vascular Surgery, Qingdao Municipal Hospital, Qingdao 266011, PR China., Department of Ultrasound, Qingdao Municipal Hospital, Qingdao 266011, PR China., Department of Science and Education, Qingdao Municipal Hospital, No. 1, Jiaozhou Road, Shibei District, Qingdao 266011, Shandong Province, PR China. Electronic address: .

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. 

Retracted: No

PubPeer comments:
 Figs 1k,l; 2a.b; 3c,d.  I am intrigued by the absence of background on these WBs, and the way the bands remain perfectly linear and retain the shape of the protein blobs as they move up the gels (and between blots). Is it possible for the authors to upload scans of the original gels? Numerous other papers have Figures which display the same qualities, hence my curiosity. I compiled a montage:  , 
 #1 Perhaps I am missing something about these Western Blots. If I understand the Figures correctly, each one contains a loading-control band (GAPDH), but the substantive bands are seem to be from separate gels, with weight markers of unknown origin at the left. How does this work with a single loading control? WBs of a very similar design and appearance can be found in [Li et al (2020)] .   , 
 The values in Tables 1 and 2 are remarkably similar.  The values in Tables 3 and 4 are also remarkably similar.  ,