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. 2018 Jan;15(1):338-344.
doi: 10.3892/etm.2017.5355. Epub 2017 Oct 23.

MicroRNA-24 inhibits the proliferation and migration of endothelial cells in patients with atherosclerosis by _targeting importin-α3 and regulating inflammatory responses

Ye Zheng  1   2 , Yongxing Li  2 , Gang Liu  3 , Xiangqian Qi  4 , Xufen Cao  2
Affiliations

MicroRNA-24 inhibits the proliferation and migration of endothelial cells in patients with atherosclerosis by _targeting importin-α3 and regulating inflammatory responses

Ye Zheng et al. Exp Ther Med. 2018 Jan.

Abstract

The aim of the present study was to measure the level of microRNA (miRNA or miR)-24 in the serum of patients with atherosclerosis and to investigate the effect of miR-24 on the expression of importin-α3 and tumor necrosis factor (TNF)-α, as well as the proliferation and migration of vascular endothelial cells. A total of 30 patients with atherosclerosis admitted to hospital between January and June 2016 were enrolled in the present study; 30 healthy subjects with a similar age range were enrolled as controls. Peripheral blood (10 ml) was collected from all participants. Human umbilical vein endothelial cells (HUVECs) were transfected with miR-24 mimic using Lipofectamine 2000. _targetScan was used to elucidate whether importin-α3 (KPNA4) was a _target gene of miR-24. Expression levels of miR-24 and mRNAs were measured using reverse transcription-quantitative polymerase chain reaction, and protein expression was determined using western blotting. Cell Counting Kit 8 assay was used to assess the proliferation of HUVECs, and a Transwell assay was performed to detect the migration of HUVECs. Expression of miR-24 in peripheral blood from patients with atherosclerosis was significantly lower when compared with healthy subjects (P<0.05). Overexpression of miR-24 was demonstrated to significantly inhibit the transcription and translation of the importin-α3 gene (P<0.05) and negatively regulate the expression of endothelial inflammatory factor TNF-α (P<0.05). Furthermore, overexpression of miR-24 significantly inhibited the proliferation and migration of HUVECs (P<0.05), and miR-24 knockdown significantly promoted these processes (P<0.05). The results of the present study suggest that miR-24 exerts its effect in atherosclerosis by blocking the nuclear factor-κB signaling pathway, regulating inflammation in endothelial cells, and inhibiting the proliferation and migration of vascular endothelial cells.

Keywords: atherosclerosis; importin-α3; microRNA.

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Figures

Figure 1.
Figure 1.
Expression of miR-24 in peripheral blood from healthy control subjects and patients with atherosclerosis. Expression of miR-24 was determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05 vs. control. miR, microRNA.
Figure 2.
Figure 2.
Bioinformatics prediction of direct interactions between miR-24 and importin-α3 using _targetScan (_targetscan.org). miR, microRNA.
Figure 3.
Figure 3.
Effect of miR-24 on the expression of importin-α3 in HUVECs. (A) Relative expression of miR-24, (B) importin-α3 mRNA and (C and D) importin-α3 protein in HUVECs from the negative control and miR-24 mimic groups. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of miR-24 and importin-α3 mRNA, and western blotting was employed to measure importin-α3 protein expression. *P<0.05 vs. negative control group. miR, microRNA; HUVECs, human umbilical vein epithelial cells.
Figure 4.
Figure 4.
Effect of miR-24 on the expression of TNF-α in HUVECs. (A) Relative expression of TNF-α mRNA in HUVECs of negative control and miR-24 mimic groups. (B and C) Relative expression of TNF-α protein in HUVECs of negative control and miR-24 mimic groups. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of TNF-α mRNA, and western blotting was employed to measure TNF-α protein expression. *P<0.05 vs. negative control group. miR, microRNA; TNF, tumor necrosis factor; HUVECs, human umbilical vein epithelial cells.
Figure 5.
Figure 5.
Proliferation of HUVECs at 24, 48, and 72 h after transfection with miR-24 mimics or inhibitors. Cell Counting Kit-8 assay was used to determine the proliferation of the cells. Absorbance of each well was measured at 450 nm with a microplate reader and cell proliferation curves were plotted. *P<0.05 vs. negative control group. miR, microRNA; HUVECs, human umbilical vein epithelial cells.
Figure 6.
Figure 6.
Effect of miR-24 on the migration ability of HUVECs. Transwell assay was used to determine the migration ability of the cells. (A) Representative images the number of migrated cells in the lower chamber (magnification, ×100) and (B) subsequent relative analysis. *P<0.05 vs. negative control group. miR, microRNA; HUVEC, human umbilical vein epithelial cell.
Figure 7.
Figure 7.
Fluorescence values of HEK293T cells transfected with WT or mutant 3′-untranslated region DNA sequences of importin-α3 and miR-24 mimic. A dual luciferase reporter assay was used to evaluate the interaction between miR-24 and importin-α3. *P<0.05 vs. NC. WT, wild type; miR, microRNA; NC, negative control.
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