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. 2020 Mar 6:19:84-96.
doi: 10.1016/j.omtn.2019.10.034. Epub 2019 Nov 12.

Knockdown of GAS5 Inhibits Atherosclerosis Progression via Reducing EZH2-Mediated ABCA1 Transcription in ApoE-/- Mice

Xiang-Dong Meng  1 , Hua-Hong Yao  1 , Li-Min Wang  1 , Min Yu  1 , Sheng Shi  1 , Zhong-Xiang Yuan  1 , Jian Liu  2
Affiliations

Knockdown of GAS5 Inhibits Atherosclerosis Progression via Reducing EZH2-Mediated ABCA1 Transcription in ApoE-/- Mice

Xiang-Dong Meng et al. Mol Ther Nucleic Acids. .

Abstract

Atherosclerosis is a disorder occurring in the large arteries and the primary cause of heart diseases. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) in atherosclerosis. This study aims to clarify the potential effects of lncRNA growth arrest-specific 5 (GAS5) on cholesterol reverse-transport and intracellular lipid accumulation in atherosclerosis. GAS5 was mainly localized in the nucleus and highly expressed in the human monocytic leukemia cell line (THP-1) macrophage-derived foam cells in coronary heart disease. Overexpressed GAS5 increased THP-1 macrophage lipid accumulation. Of note, GAS5 can inhibit the expression of ATP-binding cassette transporter A1 (ABCA1) by binding to enhancer of zeste homolog 2 (EZH2). Overexpression of EZH2 reduced cholesterol efflux and ABCA1 expression. EZH2 promoted triple methylation of lysine 27 (H3K27) in the ABCA1 promoter region. Subjected to overexpressed GAS5, overexpressed EZH2, or downregulated ABCA1, the Apolipoprotein E (ApoE)-/- mice with atherosclerosis showed increased total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE), low-density lipoprotein (LDL) levels, aortic plaque, and lipid accumulation, accompanied by reduced high-density lipoprotein (HDL) level and cholesterol outflow. Altogether, knockdown of GAS5 can potentially promote reverse-transportation of cholesterol and inhibit intracellular lipid accumulation, ultimately preventing the progression of atherosclerosis via reducing EZH2-mediated transcriptional inhibition of ABCA1 by histone methylation.

Keywords: ABCA1; EZH2; GAS5; THP-1 macrophage; atherosclerosis; cholesterol; epigenetics; long non-coding RNA.

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Figures

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Graphical abstract
Figure 1
Figure 1
GAS5 Is Expressed at a High Level in THP-1 Macrophage-Derived Foam Cells (A) Relative expression of GAS5 in THP-1 macrophage-derived foam cells and THP-1 macrophages detected using qRT-PCR. *p < 0.05 versus the THP-1 group; data were compared using unpaired t test. (B) Subcellular localization of GAS5 in THP-1 macrophage-derived foam cells detected using FISH assay (×1,000). Cellular experiments were repeated 3 times.
Figure 2
Figure 2
Overexpression of GAS5 Inhibits Cholesterol Efflux but Promotes Lipid Accumulation in THP-1 Macrophage-Derived Foam Cells (A) Transfection efficiency of GAS5 in each group determined using qRT-PCR. (B) Effect of GAS5 on the cholesterol efflux of cells detected by liquid scintillation counter. (C) Effect of GAS5 on intracellular lipid accumulation examined using oil red O staining (×400). *p < 0.05 versus the sh-NC group; #p < 0.05 versus the pLV-EGFP-N group. All cellular experiments were repeated 3 times. Comparison among multiple groups was analyzed using one-way ANOVA.
Figure 3
Figure 3
GAS5 Inhibits the Transcriptional Expression of ABCA1 by Binding to EZH2 (A) Interaction between GAS5 and EZH2 verified by RNA pull-down assay. (B) The enrichment of GAS5 by EZH2 assessed using RIP assay. (C) The effect of GAS5 on ABCA1 gene transcriptional expression assessed using qRT-PCR. (D) Transfection efficiency of sh-EZH2 and the effect of EZH2 on the transcriptional expression of ABCA1 gene determined using qRT-PCR. (E) Expression of EZH2 enriched by ABCA1 detected using ChIP. (F) ABCA1 expression enriched by H3K27me3 detected using ChIP assay. *p < 0.05 versus the IgG group or the sh-NC group; #p < 0.05 versus the pLV-FGFP-N group. Cellular experiment was repeated 3 times. Comparison between two groups was analyzed using t test and comparison among multiple groups was analyzed using one-way ANOVA.
Figure 4
Figure 4
GAS5 Plays a Role in THP-1 Macrophage-Derived Foam Cells by Recruiting EZH2 to Mediate ABCA1 Histone Trimethylation (A) Transfection efficiency of ABCA1 detected by qRT-PCR; the data were compared using unpaired t test. (B) The effects of GAS5, EZH2, and ABCA1 on reverse cholesterol transport function of THP-1 macrophage-derived foam cells detected by cholesterol efflux assay; one-way ANOVA was used for data analysis among multiple groups. (C) The effects of GAS5, EZH2, and ABCA1 on lipid accumulation in THP-1 macrophage-derived foam cells examined by oil red O staining (×400). *p < 0.05 versus the pLV-EGFP-N group; #p < 0.05 versus the pLV-EGFP-GAS5 group; &p < 0.05 versus the sh-GAS5 group.
Figure 5
Figure 5
GAS5 Downregulation Prevents Atherosclerosis Progression in ApoE−/− Mice (A) Transfection efficiency of GAS5 in each group detected by qRT-PCR and (B) transfection efficiency of sh-ABCA1 in each group detected by RT-qPCR. (C) Oil red O staining analysis of aortic plaque formation in C57BL/6J and ApoE−/−mice. (D) H&E staining analysis of aortic plaque formation in C57BL/6J and ApoE−/− mice (×400). (E) Serum cholesterol efflux in ApoE−/− mice with atherosclerosis after oe-GAS5, sh-GAS5, or sh-ABCA1 treatment. (F) Oil red O staining analysis of aortic plaque formation in ApoE−/− mice with atherosclerosis after oe-GAS5, sh-GAS5, or sh-ABCA1 treatment. (G) H&E staining analysis of aortic plaque formation in ApoE−/− mice with atherosclerosis after oe-GAS5, sh-GAS5, or sh-ABCA1 treatment (×400). *p < 0.05 versus the pLV-EGFP-N group; #p < 0.05 versus the sh-NC group; &p < 0.05 versus sh-ABCA1 group. The experiment was repeated three times. Comparison between two groups was analyzed using t test and comparison among multiple groups was analyzed using one-way ANOVA.
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