miR-133b Downregulation Reduces Vulnerable Plaque Formation in Mice with AS through Inhibiting Macrophage Immune Responses - PubMed (original) (raw)

miR-133b Downregulation Reduces Vulnerable Plaque Formation in Mice with AS through Inhibiting Macrophage Immune Responses

Cheng-Gen Zheng et al. Mol Ther Nucleic Acids. 2019.

Abstract

Atherosclerosis (AS) is a chronic inflammatory disease characterized by accumulating deposition of lipids in the arterial intima. Notably, macrophages participate centrally in the pathogenesis of this deadly disease. In this study, we established AS mouse models in order to investigate the effect of microRNA-133b (miR-133b) on vulnerable plaque formation and vascular remodeling in AS and explore the potential functional mechanisms. The expression of miR-133b was altered or the Notch-signaling pathway was blocked in the AS mouse models in order to evaluate the proliferation, migration, and apoptosis of macrophages. It was observed that miR-133b was upregulated in AS, which might target MAML1 to regulate the Notch-signaling pathway. AS mice with downregulated miR-133b or inhibited Notch-signaling pathway presented with a reduced AS plaque area, a decreased positive rate of macrophages, and an increased positive rate of vascular smooth muscle cells. Moreover, Notch-signaling pathway blockade or miR-133b downregulation inhibited the macrophage viability and migration and accelerated the apoptosis. This study provides evidence that downregulated miR-133b expression may inhibit the immune responses of macrophages and attenuate the vulnerable plaque formation and vascular remodeling in AS mice through the MAML1-mediated Notch-signaling pathway, highlighting miR-133b as a novel therapeutic target for AS.

Keywords: Notch-signaling pathway; atherosclerosis; macrophage; microRNA-133b; plaque formation.

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

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Graphical abstract

Figure 1

Figure 1

miR-133b Affects AS by Targeting MAML1 and Regulating the Notch-Signaling Pathway (A) Expression heatmap of 10 differential miRNAs in GEO:

GSE26555

. The abscissa represents sample number while the ordinate represents differential miRNAs. The upright histogram is color gradation, where each rectangle corresponds to one sample expression; red indicates high expression while green indicates low expression. (B) Comparison of target genes of miR-133b from TargetScan, miRDB, and DIANA. AS, atherosclerosis; miR-133b, microRNA-133b; MAML1, mastermind-like 1.

Figure 2

Figure 2

Downregulated miR-133b Alleviates Pathological Changes of AS Mouse Models (400×) Black arrow indicates toward typical atheromatous plaque. AS, atherosclerosis; miR-133b, microRNA-133b.

Figure 3

Figure 3

Downregulated miR-133b Increases the Positive Rate of Macrophages and Decreases the Positive Rate of VSMCs in the AS Mouse Models (A) The immunohistochemical staining of CD68 in each group (scale bar, 25 μM). (B) The immunohistochemical staining of α-SMA in each group (scale bar, 25 μM). AS, atherosclerosis; VSMC, vascular smooth muscle cell; miR-133b, microRNA-133b.

Figure 4

Figure 4

MAML1 Is a Target Gene of miR-133b (A) Binding sites of miR-133b to MAML1 based on bioinformatics prediction. (B) Luciferase activity of MAML1-WT and MAML1-Mut in the miR-133b agomir and miR-133b-NC groups. *p < 0.05, compared with the MAML1-WT + miR-133b agomir-NC group; the measurement data were presented as the mean ± SD; the experiment was repeated 3 times. miR-133b, microRNA-133b; NC, negative control; Mut, mutant; WT, wild-type; MAML1, mastermind-like 1.

Figure 5

Figure 5

Downregulated miR-133b Inhibits Macrophage Growth and Proliferation by CCK-8 Assay *p < 0.05, compared with the blank group; #p < 0.05, compared with the AS group;the measurement data were presented as the mean ± SD; the experiment was repeated 3 times; OD, optical density; AS, atherosclerosis; miR-133b, microRNA-133b; NC, negative control; CCK-8, cell counting kit-8.

Figure 6

Figure 6

Downregulation of miR-133b Inhibits Macrophage Migration by Scratch Test (A) Scratch test results of mouse macrophages in different groups. (B) Quantitative analysis for migration rate of mouse macrophages in different groups. *p < 0.05, compared with the blank group; #p < 0.05, compared with the AS group; the measurement data were presented as the mean ± SD; the experiment was repeated 3 times. AS, atherosclerosis; miR-133b, microRNA-133b.

Figure 7

Figure 7

Downregulation of miR-133b Promotes Macrophage Apoptosis by Flow Cytometry (A) Flow cytometric cell apoptosis of mouse macrophages in different groups. (B) Quantitative analysis for apoptosis rate of mouse macrophages in different groups. *p < 0.05, compared with the blank group; #p < 0.05, compared with the AS group; the measurement data were presented as the mean ± SD; the experiment was repeated 3 times. AS, atherosclerosis; miR-133b, microRNA-133b.

Figure 8

Figure 8

Downregulation of miR-133b Induces Increased MAML1 Expression and Inactivated Notch-Signaling Pathway (A) Relative expressions of miR-133b, Notch1, Jagged-1, DLL4, Bax, Bcl-2, and Caspase-3, as detected by qRT-PCR. (B) Gray values of MAML1, Notch1, Jagged-1, DLL4, Bax, Bcl-2, and Caspase-3, as determined by western blot analysis. (C) Statistical analysis of the gray values in (B). *p < 0.05, compared with the blank group; #p < 0.05, compared with the AS group; the measurement data were presented as the mean ± SD; the experiment was repeated 3 times. miR-133b, microRNA-133b; MAML1, mastermind-like 1; DLL4, Delta-like ligand 4; Bcl-2, B cell lymphoma-2; AS, atherosclerosis.

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