MiR-27b attenuates mitochondrial oxidative stress and inflammation in endothelial cells

Abstract

MiR-27b is highly expressed in endothelial cells (EC) but its function in this context is poorly characterized. This study aims to investigate the effect of miR-27b on inflammatory pathways, cell cycle, apoptosis, and mitochondrial oxidative imbalances in immortalized human aortic endothelial cells (teloHAEC), human umbilical vein endothelial cells (HUVEC), and human coronary artery endothelial cells (HCAEC) exposed to TNF-α. Treatment with TNF-α downregulates the expression of miR-27b in all EC lines, promotes the activation of inflammatory pathways, induces mitochondrial alteration and reactive oxygen species accumulation, fostering the induction of intrinsic apoptosis. Moreover, miR-27b mimic counteracts the TNF-α-related cytotoxicity and inflammation, as well as cell cycle arrest and caspase-3-dependent apoptosis, restoring mitochondria redox state, function, and membrane polarization. Mechanistically, hsa-miR-27b-3p targets the 3'untranslated regions of FOXO1 mRNA to downregulate its expression, blunting the activation of the Akt/FOXO1 pathway. Here, we show that miR-27b is involved in the regulation of a broad range of functionally intertwined phenomena in EC, suggesting its key role in mitigating mithochondrial oxidative stress and inflammation, most likely through targeting of FOXO1. Overall, results reveal for the first time that miR-27b could represent a possible target for future therapies aimed at improving endothelial health.

Keywords: Apoptosis; Endothelial dysfunction; Inflammation; Mitochondria; hsa-miR-27b-3p.

Graphical abstract

Fig. 1

TNF-α and hsa-miR-27b-3p (miR-27b) impact on EC. Hsa-miR-27b-3p (miR-27b) expression, measured by qRT-PCR and normalized with U6 as endogenous control, in (A,B) TeloHAEC, (E,F) HUVEC and (I,J) HCAEC cells treated for 8h with 40 ng/mL TNF-α or transfected with 20 nM mimic Negative Control (miR-NC) and miR-27b mimic (miR-27b). MiR levels are reported as floating bars with line representing the mean ± SD. EC viability and LDH release were evaluated after 8h treatment with 40 ng/mL TNF-α on (C,D) TeloHAEC, (G,H) HUVEC and (K,L) HCAEC transfected or not with miR-NC and miR-27b. Control cells (Ctr) were treated with corresponding volumes of HBSS-10 mM Hepes. Data are expressed as mean ± SD of n = 3 experiments. *p < 0.05 vs. Ctr; ¶p < 0.001 vs. Ctr; §p < 0.001 vs. miR-NC; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 2

TNF-α and miR-27b effects on inflammation. Evaluation of VCAM1, ICAM1, IL-6, and NO levels in (A–D) TeloHAEC, (E–H) HUVEC and (I–L) HCAEC cells exposed for 8h to 40 ng/mL TNF-α or transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were treated with corresponding volumes of HBSS-10 mM Hepes. Data are expressed as mean ± SD of n = 3 experiments. ¶p < 0.001 vs. Ctr; §p < 0.001 vs. miR-NC; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 3

Cell cycle evaluation. (A,B) Representative cell cycle analysis and cropped blots with relative immunoblotting analysis of (C,D) cyclin D1 and (E,F) cyclin E1 protein levels in teloHAEC cells transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were dealt with correponding volumes of HBSS-10 mM Hepes. Lane 1 = Ctr; lane 2 = miR-NC; lane 3 = miR-NC+TNF-α; lane 4 = miR-27b; lane 5 = miR-27b+TNF-α; M = molecular weight markers. Western blotting results are expressed as arbitrary units (AU). §p < 0.001 vs. miR-NC; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 4

Mitochondrial oxidative stress. (A–C) Representative fluorescent images and FACS analysis of mitochondrial ROS detection, (D) MDA, (E) GSH/GSSG and (F) NAD⁺/NADH content and cropped blots with relative immunoblotting analysis of (G,H) p66^Shc, (I,J) COX-IV, and (K,L) acetylated-SOD2/SOD2 protein levels in teloHAEC transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were dealt with corresponding volumes of HBSS-10 mM Hepes. Scale bars = 100 μm. Lane 1 = Ctr; lane 2 = miR-NC; lane 3 = miR-NC+TNF-α; lane 4 = miR-27b; lane 5 = miR-27b+TNF-α; M = molecular weight markers. Western blotting results are expressed as arbitrary units (AU). §p < 0.001 vs. miR-NC; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 5

Mitochondrial membrane depolarization. (A–C) Representative fluorescent images and FACS analysis of mitochondrial membrane potential, (D,E) mitochondrial permeability transition pore and (F) ATP content in teloHAEC transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were treated with corresponding volumes of HBSS-10 mM Hepes. Scale bars = 100 μm. Data are expressed as mean ± SD of n = 3 experiments. §p < 0.001 vs. miR-NC; •p < 0.05 vs. miR-NC+TNF-α; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 6

Mitochondrial damage. (A–C) Representative fluorescent images and FACS analysis of mitophagy (red) and lysosome (green) dyes and (D–F) green-stained intact mitochondria in teloHAEC transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were treated with corresponding volumes of HBSS-10 mM Hepes. Scale bars = 100 μm. Data are expressed as mean ± SD of n = 3 experiments. *p < 0.05 vs. miR-NC; §p < 0.001 vs. miR-NC; †p < 0.01 vs. miR-NC+TNF-α. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Mitochondrial apoptosis. (A–C) Representative fluorescent images and FACS analysis of Nucview 488 and Mitoview 633 staining, (F,G) dot plots and analysis of annexin V-FITC and PI-staining and cropped blots with relative immunoblotting analysis of (D,E) caspase-3 and (H,I) Bax/Bcl-2 protein levels in teloHAEC transfected with miR-NC and miR-27b before TNF-α stimulation. Control cells (Ctr) were treated with corresponding volumes of HBSS-10 mM Hepes. Western blotting results are expressed as arbitrary units (AU). Lane 1 = Ctr; lane 2 = miR-NC; lane 3 = miR-NC+TNF-α; lane 4 = miR-27b; lane 5 = miR-27b+TNF-α; M = molecular weight markers. Cell viability/death was assessed by flow cytometry where at least 10,000 events were acquired. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Scale bars = 100 μm §p < 0.001 vs. miR-NC; •p < 0.05 vs. miR-NC+TNF-α; †p < 0.01 vs. miR-NC+TNF-α.

Fig. 8

MiR-27b-FOXO1 interaction. (A) Alignment of hsa-miR-27b-3p with two regions (426–432, 2000–2006) of FOXO1 3′-UTR from miRNA-mRNA integration analysis using miRNA target prediction tools, as TargetScan and miRDB. (B) Schematic diagram of luciferase reporter plasmid containing 3′-UTR target sequence (region 350–550) of FOXO1. P: promoter, LUC: Luciferase. Representative analysis of (C) mRNA and (D,E) cropped blots with relative immunoblotting analysis of FOXO1 and (F,G) phospho-Akt/Akt protein levels in teloHAEC transfected with miR-NC and miR-27b before TNF-α stimulation. Western blotting results are expressed as arbitrary units (AU). Lane 1 = Ctr; lane 2 = miR-NC; lane 3 = miR-NC+TNF-α; lane 4 = miR-27b; lane 5 = miR-27b+TNF-α; M = molecular weight markers. (H) The relative luciferase activity in teloHAEC co-transfected with luciferase reporter plasmid, containing the region 350–550 of FOXO1 3′-UTR, and miR-NC or miR-27b. Control cells (Ctr) were treated with Corresponding volumes of HBSS-10 mM Hepes. Data are expressed as mean ± SD of n = 3 experiments. *p < 0.05 vs. miR-NC; ‡p < 0.01 vs. miR-NC; §p < 0.001 vs. miR-NC; •p < 0.05 vs. miR-NC+TNF-α; †p < 0.01 vs. miR-NC+TNF-α.