Abstract
Background: Atherosclerosis, a cardiovascular disease caused by several risk factors, is one of the major contributors to mortality in developed countries. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of cyclin-dependent kinase inhibitor 2B antisense noncoding RNA (CDKN2B-AS1) and CDKN2B in progression of atherosclerosis. Methods: Initially, we quantified the expression ofCDKN2B-AS1 in atherosclerotic plaque tissues and THP-1 macrophage-derived foam cells by qRT-PCR and western blot, followed by a mouse model establishment of atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice. Subsequently, lipid uptake, lipid accumulation and macrophage reverse cholesterol transport (mRCT) were detected to explore the contributory role of CDKN2B-AS1 in the progression of atherosclerosis. Furthermore, we performed RIP and ChIP to identify the interaction among CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2) and CDKN2B. Results: Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment. CDKN2B-AS1 showed high expression in atherosclerosis and CDKN2B showed low expression. Silencing of CDKN2B-AS1 decreased the ability of lipid uptake and intracellular lipid accumulation and promoted mRCT in THP-1 macrophage-derived foam cells and mouse models. EZH2 and CTCF were found to bind to CDKN2B promoter region. A RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in CDKN2B promoter region and subsequently inhibited CDKN2B transcription by accelerating histone methylation. Conclusion: The key findings of this study collectively propose silencing of CDKN2B-AS1 inhibits atherosclerotic plaque formation and promotes mRCT in atherosclerosis by regulating CDKN2B promoter, which can be explored to treat this vascular disease. Funding: This study was supported by the General Projects of National Natural Science Foundation of China — the Function and Regulation Mechanism of ETBR in Aortic Endothelium and Smooth Muscle Cells in Pregnancy Induced Hypertension (No. 81871187), the Regional Projects of the National Natural Science Foundation of China — Correlation Analysis Between Endothelin Receptor and Pregnancy Induced Hypertension and Abnormal Blood Pressure Regulation (No. 81460239), and the Natural Science Foundation Project of Ningxia Province — Correlation Analysis Between Anastomotic Regional Blood Flow Velocity and Intimal Hyperplasia after Revascularization (No. NZ17195). Declaration of Interest: The authors have declared that no competing interests exist. Ethical Approval: This research study was conducted with the approval of the ethics committee of Qingdao Municipal Hospital. All patients participating in the study signed informed written consent documentation, according to Helsinki Declaration. The animal experiment procedures were performed in accordance with strict protocols approved by the Institutional Animal Care and Use Committee.
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