Propofol Upregulates MicroRNA-30b to Inhibit Excessive Autophagy and Apoptosis and Attenuates Ischemia/Reperfusion Injury In Vitro and in Patients

Abstract

Evidence reveals that propofol protects cells via suppressing excessive autophagy induced by hypoxia/reoxygenation (H/R). Previously, we found in a genome-wide microRNA profile analysis that several autophagy-related microRNAs were significantly altered during the process of H/R in the presence or absence of propofol posthypoxia treatment (P-PostH), but how these microRNAs work in P-PostH is still largely unknown. Here, we found that one of these microRNAs, microRNA-30b (miR-30b), in human umbilical vein endothelial cells (HUVECs) was downregulated by H/R treatment but significantly upregulated by 100 M propofol after H/R treatment. miR-30b showed similar changes in open heart surgery patients. By dual-luciferase assay, we found that Beclin-1 is the direct target of miR-30b. This conclusion was also supported by knockdown or overexpression of miR-30b. Further studies showed that miR-30b inhibited H/R-induced autophagy activation. Overexpression or knockdown of miR-30b regulated autophagy-related protein gene expression in vitro. To clarify the specific role of propofol in the inhibition of autophagy and distinguish the induction of autophagy from the damage of autophagy flux, we used bafilomycin A1. LC3-II levels were decreased in the group treated with propofol combined with bafilomycin A1 compared with the group treated with bafilomycin A1 alone after hypoxia and reoxygenation. Moreover, HUVECs transfected with Ad-mCherry-GFP-LC3b confirmed the inhibitory effect of miR-30b on autophagy flux. Finally, we found that miR-30b is able to increase the cellular viability under the H/R condition, partially mimicking the protective effect of propofol which suppressed autophagy via enhancing miR-30b and targeting Beclin-1. Therefore, we concluded that propofol upregulates miR-30b to repress excessive autophagy via targeting Beclin-1 under H/R condition. Thus, our results revealed a novel mechanism of the protective role of propofol during anesthesia. Clinical Trial Registration Number. This trial is registered with ChiCTR-IPR-14005470. The name of the trial register: Propofol Upregulates MicroRNA-30b to Repress Beclin-1 and Inhibits Excessive Autophagy and Apoptosis.

Figure 1

The change of myocardial enzymes during perioperative period. (a) The change of plasma TNT-HS between two groups in the perioperative period. (b) The change of plasma CK-MB between two groups in the perioperative period. Mean ± SD of 3 independent trials. ^∗p < 0.05, vs. the control group. Abbreviations: T₀: before anesthesia induction; T₁: 15 min before cardiopulmonary bypass; T₂: 15 min after cardiac arrest; T₃: 10 minutes after heart restated beating; T₄: 1 h after heart restated beating; T₅: 24 h after operation; TNT-HS: sensitive troponin T; CK-MB: creatine kinase isoenzyme muscle/brain.

Figure 2

Propofol inhibits autophagy in H/R (I/R) models in vitro or in vivo. (a–d) The PI3K inhibitor 3-methyladenine (10 mM) was used to ascertain the activation of autophagy. ^∗p < 0.05, vs. the H/R group. ^∗∗p < 0.01, vs. the H/R group. ^∗∗∗p < 0.001, vs. the H/R group. (e) HUVECs were transduced with Ad-mRFP-GFP-LC3B and then treated with 100 μmol/L propofol or 10 mmol/L 3-MA after H/R. Representative images of fluorescent LC3 puncta. Yellow puncta: red puncta overlaid with green puncta and red puncta: autolysosomes. Scale bar = 20 μm. (f–h) The cells were postconditioned at the very onset of reoxygenation with increasing concentrations of propofol (0–150 μmol/L) for 4 h after 12 h of hypoxia. Expression of autophagy-related proteins in control group, H/R injury group, and propofol posthypoxia treatment groups. ^∗p < 0.05, vs. the H/R group. ^∗∗p < 0.01, vs. the H/R group. (i–j) The level of autophagy-related proteins Beclin-1 was examined in the control group (patients treated with midazolam and sevoflurane during reperfusion) and propofol group (patients treated with propofol and sevoflurane during reperfusion). ^∗p < 0.05, T₃ in the propofol group vs. T₃ in the control group. ^#p < 0.05, T₃ in the propofol group vs. T₂ in the propofol group. Mean ± SD of 3 independent trials. Abbreviations: H/R: hypoxia/re-oxygenation; P: propofol; 3-MA: 3-methyladenine; Ctrl or C: the control group; T₁: 15 min before cardiopulmonary bypass; T₂: 15 min after cardiac arrest; T₃: 10 minutes after heart restated beating.

Figure 3

Propofol induces miR-30b expression in H/R (I/R) models in vitro or in vivo. (a) The expression of miR-30b in each group by real-time PCR. ^∗∗p < 0.01, vs. the control group. (b) The different expression of miR-30b between the control group and the propofol group by real-time PCR. ^∗∗∗p < 0.001, T₃ in the propofol group vs. T₃ in the control group. (c) The different expression of miR-30b in each group by real-time-PCR. ^∗∗∗p < 0.001, T₃ in the propofol group vs. T₂ in the propofol group.

Figure 4

Beclin-1 is a direct target of miR-30b. (a) Diagram of the miR-30b pairing sequence in 3′UTR of Beclin-1. The matched base pairs are connected by a vertical line. Beclin-1 3′UTR relevant fragments were inserted downstream of the firefly luciferase gene of the pmirGLO vector. (b) The different expression of relative fluorescence intensity in each group by dual-luciferase reporter gene assay. The 293T cells were cotransfected with 3′UTR Beclin-1 plasmid and each different miRNA, and the relative fluorescence intensity of the samples was calculated. ^∗p < 0.05, vs. the hsa-miR-30b-5p group. p > 0.05, the NC group vs. the hsa-miR-30b-5p inhibitor group. p > 0.05, the NC inhibitor group vs. the hsa-miR-30b-5p inhibitor group. (c) The different expression of relative fluorescence intensity in each group by dual-luciferase reporter gene assay. The 293T cells were cotransfected with mut3′UTR Beclin-1 plasmid and each different miRNA, and the relative fluorescence intensity of the samples was calculated. p > 0.05, the NC group vs. the hsa-miR-30b-5p group. p > 0.05, the blank group vs. the hsa-miR-30b-5p group. (d–e) The expression of autophagy-related protein Beclin-1 in blank and transfecting cells with miR-30b, miR-30b control, miR-30b inhibitor, and miR-30b inhibitor control groups without H/R treatment. ^∗p < 0.05, vs. the hsa-miR-30b-5p group. Mean ± SD of 3 independent trials. Abbreviations: miR-30b: hsa-miR-30b-5p; NC: the negative control.

Figure 5

miR-30b suppresses H/R-induced autophagy activation. (a–d) The HUVECs were transfected with each different miRNA. The different expressions of Beclin-1, LC3-II, and p62 in each group. (e–f) The expressions of GAPDH and LC3-II in the normal group, the H/R group, and 100 μmol/L propofol or 10 μM bafilomycin A1 H/R treatment group. ^∗p < 0.05, vs. the Baf+p+H/R group. ^#p < 0.05, the control group vs. the Baf+p+H/R group. (g) The HUVECs were transfected with Ad-mCherry-GFP-LC3B and observed under a fluorescence microscope. Yellow puncta: red puncta overlaid with green puncta and red puncta: autolysosomes. Scale bar = 20 μm. Mean ± SD of 3 independent trials. Abbreviations: 3-MA: 3-methyladenine; HUVECs: human umbilical vein endothelial cell; P: propofol; H/R: hypoxia/reoxygenation; miR-30b: hsa-miR-30b-5p; NC: the negative control; Baf: bafilomycin A1.

Figure 6

MicroRNA-30b represses H/R induced autophagy and promotes cell survival. The cells were transfected with miR-30b, NC, miR-30b inhibitor, and NC inhibitor and treated with 12 h of hypoxia and 4 h of reoxygenation. (a–c) The expression of Bcl-2, Bax in control, hypoxia reoxygenation injury group (H/R), 100 μmol/L propofol posthypoxia treatment group (P), and transfecting microRNAs groups were examined. ^∗p < 0.05, vs. the H/R group. (d–f) Cell viability was determined by CCK-8 assay, as previously described. Values are presented as the percentage of viable cells. ^∗p < 0.05, compared with the H/R group. ^∗∗p < 0.01, compared with the H/R group. ^∗∗∗p < 0.001, compared with the H/R group.