Propofol-induced MiR-20b expression initiates endogenous cellular signal changes mitigating hypoxia/re-oxygenation-induced endothelial autophagy in vitro

Abstract

Certain miRNAs can attenuate hypoxia/re-oxygenation-induced autophagic cell death reported in our previous studies, but how these miRNAs regulate the autophagy-related cellular signaling pathway in preventing cell death is largely unknown. In the current study, the autophagy-related miRNAs of hsa-miR-20b were investigated in an in vitro model of hypoxia/re-oxygenation-induced endothelial autophagic cell death. Of these, miR-20b was found to be the most important miRNA which targeted on the key autophagy kinase ULK1 and inhibited hypoxia/re-oxygenation injury-induced autophagy by decreasing both autophagosomes and LC3I to II transition rate and P62 degradation. These processes were reversed by the transfection of an miR-20b inhibitor. Re-expression of ULK1 restores miR-20b-inhibited autophagy. Propofol, a commonly used anesthetic, promoted miR-20b and METTL3 expression and attenuated endothelial autophagic cell death. The inhibited endogenous expression of miR-20b or silenced METTL3 diminished the protective effect of propofol and accentuated autophagy. Additionally, METTL3 knockdown significantly inhibited miR-20b expression but up-regulated pri-miR-20b expression. Together, our data shows that propofol protects against endothelial autophagic cell death induced by hypoxia/re-oxygenation injury, associated with activation of METTL3/miR-20b/ULK1 cellular signaling.

Fig. 1. miR-20b targets ULK1 and regulates its expression.

a Luciferase reporter constructs. The 3′UTR (1–1801 bp) of ULK1 was inserted downstream of the firefly luciferase gene of the pmirGLO vector, named pmirGLO-ULK1. MRE denotes miRNA response element. b Luciferase reporter assay of the interaction between miR-20b and the predicted MRE in HUVECs. Each pmirGLO-ULK1 was co-transfected with negative control (NC), miR-20b mimics (20b), inhibitor NC (IN NC), or miR-20b inhibitor (IN 20b) into HUVECs. Detection of the luciferase activity was done after 24 h. The firefly luciferase activity was normalized to Renilla. c, d qPCR was performed to detect the expression of miR-20b and ULK1 in HUVECs, respectively. e, f HUVECs lysates were prepared and subjected to western blot analysis by using anti-ULK1 antibody. Data were mean ± SEM (n = 3); *p < 0.05,**p < 0.01, ***p < 0.001.

Fig. 2. Propofol suppresses H/R-induced autophagy.

a Built of the hypoxia/re-oxygenation model. The culture media was replaced with glucose and serum free DMEM. Then the HUVECs in hypoxic conditions was placed with 94% N₂, 5% CO₂, and 1% O₂ using a small enclosed chamber filled at 37 °C for 12 h. Then the medium was changed to culture media and propofol was added with different concentrations: 25 μmol/L (P25), 50 μmol/L (P50), 100 μmol/L (P100), 150 μmol/L (P150) for 4 h in normal condition. b–e The expression of ULK1, Beclin1, and LC3 was determined in normal HUVECs, H/R injury HUVECs, H/R+DMSO HUVECs, and propofol post-hypoxia treatment HUVECs. f Autophagosomes and mitochondria were probed by anti-LC3 (green) and anti-TOM20 (red) in each group as above. Bar, 10 μm. Data were from three independent experiments. g–i PCR was performed to detect the expression of miR-20b, Beclin1, and ULK1 in normal HUVECs, H/R injury HUVECs, H/R+DMSO HUVECs, and propofol-treated HUVECs with the most effective concentration of 100 μmol/L. Data were mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. miR-20b regulates H/R-induced autophagy by targeting ULK1.

a The HUVECs were transfected with negative control (NC), miR-20b mimics (20b), inhibitor NC (IN NC), miR-20b inhibitor (IN 20b) for 24 h. Then the HUVECs in hypoxic conditions were placed as above for 12 h. The medium was only replaced and placed in normal condition for 4 h after hypoxia. b–f Total proteins were extracted to detect ULK1, P62, Beclin1, LC3II/I (16KD/18KD) by using β-actin as a reference. Densitometric ratios of these proteins were quantified by using IMAGEJ. g Samples were stained with anti-LC3 (red) and anti-p62 (blue) antibodies. Bar, 10 μm. h Samples were stained with ULK1 (red) antibodies. Bar, 10 μm. i Electron microscopy was used to assess the inhibitory role of miR-20b in the H/R-induced autophagy. Bar, 1 μm. Data are mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. Re-expression of ULK1 restores autophagy inhibited by miR-20b.

Built the H/R model as above. a–d HUVECs were transfected with negative control (NC) and miR-20b mimics (20b) or co-transfected with miR-20b mimics (20b) and the constructs (ULK1-Flag) were indicated for 24 h. Using a normal group as a control. Built the H/R model with these cells. Protein samples were collected and the expression of ULK1, Flag, LC3 (16KD/18KD), and P62 was detected through western blot. β-actin was used as a reference. e Treat the cells as in a. Then the cells were processed for immunofluorescence with anti-TOM20 (red) and LC3 (green) antibodies. Bar, 10 μm. Data are mean ± SEM (n = 3); *p < 0.05, **p < 0.01,***p < 0.001.

Fig. 5. Inhibiting the expression of miR-20b reduces the inhibitory effect of propofol on autophagy.

Built the H/R model as above. a–d HUVECs were transfected with miR-20b mimics (20b) or miR-20b inhibitor (IN 20b) for 24 h. Built the H/R model with these cells and the cells were treated with or without propofol at re-oxygenation for 4 h. Divided the cells into five groups: H/R Normal+P100, H/R 20b+P100, H/R 20b+DMSO, H/R IN 20b+P100, and H/R IN 20b+DMSO. Protein samples were collected and the expression of ULK1, LC3 (16KD/18KD), and P62 was detected through western blot. e Treat the cells as in a. Then the cells were processed for immunofluorescence with P62 (blue) and LC3 (red) antibodies. Bar, 10 μm. Data were mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 6. Propofol promotes METTL3-mediated pri-miR-20b maturation.

Built the H/R model as above. a, b qPCR analyses of the level of pri-miR-20b and METTL3. c–e Western blotting analyses of DGCR8 and METTL3 levels. f, g Western blotting verified the successful knockdown of METTL3. h, i qPCR analysis of the level of pri-miR-20b and miR-20b after knockdown of METTL3. Data are shown as the mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7. METTL3 knockdown restores autophagy inhibited by propofol.

a–f Built the H/R model after knockdown of METTL3. Western blotting analysis of the autophagy-related protein and DGCR8 levels. g–l Built the H/R model after knockdown of METTL3 and treated the cells with or without propofol at re-oxygenation for 4 h. Western blotting analysis of the autophagy-related protein and DGCR8 levels. Data are shown as the mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 8. Propofol promotes cytoprotective mechanisms via miR-20b.

miR-20b expression induced by propofol promotes endogenous cytoprotective mechanisms of the METTL3/miR-20b/ULK1 regulatory “network” in hypoxia/re-oxygenation injury. METTL3: methyltransferase-like 3; DGCR8: DiGeorge critical region 8; ULK1: Unc-51-like kinase 1; mRNA: Messenger RNA; pri- miR-20b: primary microRNA-20b; pre-miR-20b: precursor microRNA-20b; miR-20b: microRNA-20b.