Silencing of long non-coding RNA Sox2ot inhibits oxidative stress and inflammation of vascular smooth muscle cells in abdominal aortic aneurysm via microRNA-145-mediated Egr1 inhibition

Abstract

Long non-coding RNAs (lncRNAs) have been largely reported to contribute to the development and progression of abdominal aortic aneurysm (AAA), a common vascular degenerative disease. The present study was set out with the aim to investigate the possible role of lncRNA Sox2ot in the development of AAA. In this study, we found that lncRNA Sox2ot and early growth response factor-1 (Egr1) were highly expressed, while microRNA (miR)-145 was poorly expressed in Ang II-induced AAA mice and oxidative stress-provoked vascular smooth muscle cell (VSMC) model. Egr1 was a potential target gene of miR-145, and lncRNA Sox2ot could competitively bind to miR-145 to upregulate Egr1 expression. Overexpression of miR-145-5p was found to attenuate oxidative stress and inflammation by inhibiting Egr1 both in vivo and in vitro, which was counteracted by lncRNA Sox2ot. Taken together, the present study provides evidence that downregulation of lncRNA Sox2ot suppressed the expression of Egr1 through regulating miR-145, thus inhibiting the development of AAA, highlighting a theoretical basis for AAA treatment.

Keywords: Egr1; MicroRNA-145; abdominal aortic aneurysm; long non-coding RNA Sox2ot; oxidative stress.

Figure 1

GEO bioinformatics analysis predicting poorly expressed miR-145 in mice with Ang II-induced AAA. (A) a heatmap of the top 10 differentially expressed miRNAs obtained from the AAA-related microarray data GSE51226 downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/); the abscissa represents sample number and the ordinate represents names of miRNAs; each small square in the figure represents the expression level of a miRNA in one sample, and the histogram in the upper right represents color grading; (B) representative images of the morphology of abdominal aorta specimens of the control ApoE^-/- mice and Ang II-induced AAA ApoE^-/- mice; (C) incidence of AAA in ApoE^-/- mice; (D) the maximum diameter of abdominal aorta in mice; (E) morphological changes of abdominal aorta in mice observed by HE staining (× 400); (F) α-SM-actin expression in SMCs in abdominal aorta determined using immunohistochemistry (× 400); (G) MOMA-2 expression in monocyte and SMCs in abdominal aorta determined using immunohistochemistry (× 400); (H) miR-145 expression measured using RT-qPCR; * p < 0.05 compared with ApoE^-/- mice; measurement data were depicted as the mean ± standard deviation; comparisons between the two groups were analyzed using an unpaired t-test; n = 10.

Figure 2

miR-145 suppresses the occurrence and progression of AAA in ApoE^-/- mice. (A) interference efficiency of miR-145 verified by RT-qPCR; (B) representative morphology images of abdominal aorta specimens in mice; (C) incidence of AAA in mice; (D) the maximum diameter of abdominal aorta in mice; (E) α-SM-actin expression in SMCs in abdominal aorta determined using immunohistochemistry (× 400); (F) CD68 expression in abdominal aorta detected using immunofluorescence staining (× 400); (G) levels of COX-2, NO, IL-1β, IL-6 and TNF-α in serum of mice measured using ELISA; (H) SOD level in serum and MDA level in abdominal aorta of mice; (I) protein levels of cleaved caspase-3, NOX4, iNOS, p47phox, collagen I and collagen III determined using Western blot analysis; * p < 0.05, vs. normal mice; # p < 0.05, vs. AAA mice injected with LV-miR-NC or LV-NC-inhibitor plasmids; measurement data were depicted as the mean ± standard deviation; comparisons among multiple groups were analyzed using one-way ANOVA followed by Turkey’s post hoc test; n = 10.

Figure 3

miR-145 suppresses the apoptosis, inflammatory reaction, and oxidative stress of VSMCs treated with Ang II. (A) the expression of the molecular marker α-SM-actin of VSMCs isolated by immunofluorescence assay (× 400); (B) miR-145 expression in VSMCs determined using RT-qPCR; (C) viability of VSMCs detected by CCK-8 assay; (D) apoptosis of VSMCs detected by flow cytometry; (E) levels of COX-2, NO, IL-1β, IL-6, and TNF-α in serum of VSMCs measured by ELISA; (F) levels of MDA and SOD in serum of VSMCs measured using kits; (G) O₂^- in VSMCs measured using DHE staining (× 400); (H) protein levels of cleaved caspase-3, NOX4, iNOS, p47phox, collagen I and collagen III in VSMCs determined using Western blot analysis; *, p < 0.05, vs. VSMCs without treatment; #, p < 0.05, vs. VSMCs treated with Ang II + LV-miR-NC or Ang II+LV-NC-inhibitor plasmids; measurement data were depicted as the mean ± standard deviation; comparisons among multiple groups were analyzed using one-way ANOVA followed by Turkey’s post hoc test; the experiment was repeated three times.

Figure 4

miR-145 is negatively regulated by lncRNA Sox2ot. (A) comparisons of predicted lncRNAs that target miR-145 in RAID database (http://www.rna-society.org/raid/index.html) and lncRNAs related to AAA reported in a prior study; (B) Sequence complementarity between lncRNA Sox2ot and miR-145 analyzed by RNA22; (C) lncRNA Sox2ot expression in abdominal aorta of normal mice and Ang II-induced AAA mice determined using RT-qPCR; (D) lncRNA Sox2ot expression in VSMCs determined by RT-qPCR; (E) miR-145 expression in VSMCs measured by RT-qPCR; (F) RIP assay with anti-Ago2, IgG, or 10% input from VSMC extracts. RNA levels in the immunoprecipitates were determined by RT-qPCR; (G) co-localization of miR-145 and lncRNA Sox2ot detected by FISH (× 400); * p < 0.05, vs. VSMCs without treatment; # p < 0.05, vs. VSMCs treated with Ang II + LV-miR-NC or Ang II + LV-NC-inhibitor plasmids; measurement data were depicted as the mean ± standard deviation; comparisons between the two groups were analyzed using an unpaired t-test, and comparisons among multiple groups were analyzed using one-way ANOVA, followed by Turkey’s post hoc test; each experiment was repeated three times.

Figure 5

LncRNA Sox2ot reverses the inhibitory roles of miR-145 in oxidative stress and inflammation of VSMCs treated with Ang II. (A) viability of VSMCs detected by CCK-8 assay; (B) apoptosis of VSMCs detected by flow cytometry; (C) levels of COX-2, NO, IL-1β, IL-6, and TNF-α in serum of VSMCs measured by ELISA; (D) levels of MDA and SOD in serum of VSMCs measured using kits; (E) ROS level in VSMCs measured using kits; (F) protein levels of cleaved caspase-3, NOX4, iNOS, p47phox, collagen I and collagen III determined using Western blot analysis; * p < 0.05, vs. VSMCs without treatment; # p < 0.05, vs. VSMCs treated with Ang II + NC-mimic, Ang II + NC-inhibitor, and Ang II + miR-145-mimic + NC plasmids; measurement data were depicted as the mean ± standard deviation; comparisons among multiple groups were analyzed using one-way ANOVA followed by Turkey’s post hoc test; the experiment was repeated three times.

Figure 6

Silencing of lncRNA Sox2ot binding miR-145 inhibits oxidative stress and inflammation of Ang II-treated VSMCs by downregulating Egr1. (A) Venn map for 119 intersection genes predicted to be regulated by miR-145 in the DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), miRmap (https://mirmap.ezlab.org/), microRNA.org (http://www.microrna.org/microrna/home.do), and starBase (http://starbase.sysu.edu.cn/index.php); (B) a PPI network using Cytoscape 3.6.0 software for target genes of miR-145 was constructed using string database (https://string-db.org/); the color represents the correlation between miR-145 and other genes, and red represent the high correlation; (C) the complementary binding sites between miR-145 and Egr1 3'-UTR predicted by StarBase; (D) Egr1 mRNA and protein levels in the abdominal aorta of normal mice and Ang II-induced AAA mice determined by RT-qPCR and Western blot analysis; (E) interaction between miR-145 and Egr1 detected by dual luciferase reporter gene assay; (F) the interaction between miR-145 and Egr1 verified by RNA pull down assay; (G) Egr1 mRNA and protein levels in VSMCs determined by RT-qPCR and Western blot analysis; (H) cell viability of VSMCs detected by CCK-8 assay; (I) apoptosis of VSMCs detected by flow cytometry; (J) levels of COX-2, NO, IL-1β, IL-6, and TNF-α in serum of VSMCs Egr1 and/or Sox2ot/miR-145 measured by ELISA; (K) levels of MDA and SOD in serum of VSMCs measured using kits; (L) ROS level in VSMCs measured by kits; (M) protein levels of cleaved caspase-3, NOX4, iNOS, p47phox, collagen I and collagen III in VSMCs determined using Western blot analysis; * p < 0.05, vs. normal mice or VSMCs without treatment; # p < 0.05, vs. VSMCs treated with Ang II + NC-mimic, Ang II + NC-inhibitor, Ang II + NC, or Ang II + si-NC plasmids; & p < 0.05, vs. VSMCs treated with Ang II + NC-mimic or Ang II + Egr1 plasmids; measurement data were depicted as the mean ± standard deviation; comparisons between the two groups were analyzed using unpaired t-test (n = 10), and comparisons among multiple groups were analyzed using one-way ANOVA, followed by Turkey’s post hoc test; the experiment was repeated three times.

Figure 7

Silencing of lncRNA Sox2ot affecting miR-145 inhibits oxidative stress and inflammation in Ang II-induced AAA mice by downregulating Egr1. (A) representative morphology images of abdominal aorta specimens from mice; (B) incidence of inducing AAA mice; (C) the maximum diameter of abdominal aorta from mice; (D) α-SM-actin expression in SMCs in abdominal aorta determined using immunohistochemistry (× 400); (E) CD68 expression in abdominal aorta detected using immunofluorescence staining (× 400); (F) levels of inflammatory factors and oxidative stress-related factors measured in serum of mice measured using ELISA; (G) SOD level in serum and MDA levels in abdominal aorta of mice; (H) protein levels of Egr1, M-CSF, MCP-1, MIP-2, ICAM-1, cleaved caspase-3, iNOS, p47phox, collagen I and collagen III determined using Western blot analysis; * p < 0.05, vs. normal mice; # p < 0.05, vs. AAA mice injected with LV-NC; & p < 0.05, vs. AAA mice injected with LV-Sox2ot; @ vs. AAA mice, injected with LV-Egr1; measurement data were depicted as the mean ± standard deviation; comparisons among multiple groups were analyzed using one-way ANOVA followed by Turkey’s post hoc test; n = 10.

Figure 8

A mechanism graph of the lncRNA Sox2ot/miR-145/Egr1 axis involved in mice with Ang II-induced AAA. LncRNA Sox2ot competitively binds to miR-145 and negatively regulates its expression, thus upregulating the expression of Egr1 and then promoting the apoptosis, inflammatory reaction, and oxidative stress of VSMCs, which ultimately accelerates the occurrence of AAA induced by Ang II.