Inhibition of long noncoding RNA HIF1A-AS2 confers protection against atherosclerosis via ATF2 downregulation

J Adv Res. 2020 Jul 31;26:123-135. doi: 10.1016/j.jare.2020.07.015. eCollection 2020 Nov.

Abstract

Introduction: In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis.

Objectives: Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation.

Methods: An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1).

Results: It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation.

Conclusion: This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

Keywords: ATCC, American Type Culture Collection; ATF2, activating transcription factor 2; Activating transcription factor; Atherosclerosis; CAD, coronary artery disease; CCK-8, cell counting kit-8; ChIP, Chromatin immunoprecipitation; DMEM, Dulbecco’s modified Eagle’s medium; ECs, endothelial cells; ELISA, enzyme linked immunosorbent assay; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HCAECs, human coronary artery endothelial cells; HE, Hematoxylin-eosin; HFD, high fat diet; HIF1A-AS2, hypoxia-inducible factor 1 alpha-antisense RNA 2; Hypoxia-inducible factor 1 alpha-antisense RNA 2; ICAM-1, intercellular adhesion molecule-1; IL-1β, interleukin-1β; IL-6, interleukin-6; IgG, immunoglobulin G; Inflammation; LDL, low-density lipoprotein; Long noncoding RNA; MCP-1, monocyte chemoattractant protein-1; ND, normal diet; PBS, phosphate buffered saline; RIP, RNA binding protein immunoprecipitation; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SMCs, smooth muscle cells; TNF-α, tumor necrosis factor-α; Transcription factor; USF1, upstream stimulatory factor 1; Upstream transcription factor 1; VCAM-1, vascular cell adhesion molecule 1; lncRNAs, long noncoding RNAs; ox-LDL, oxidized-low-density lipoprotein; sh, short hairpin RNA; si-NC, small interfering RNA-negative control.