LncRNA TUG1 alleviates sepsis-induced acute lung injury by targeting miR-34b-5p/GAB1

Abstract

Background: Sepsis-induced acute lung injury (ALI) is a clinical syndrome characterized by the injury of alveolar epithelium and pulmonary endothelial cells. This study aimed to investigate the regulation of long noncoding RNA (lncRNA) taurine up-regulated gene 1 (TUG1) in a murine ALI model and in primary murine pulmonary microvascular endothelial cells (PMVECs) stimulated with lipopolysaccharide (LPS).

Methods: Adult C57BL/6 mice were intravenously injected with or without TUG1-expressiong adenoviral vector or control vector 1 week before the establishment of ALI model. PMVECs were transfected with TUG1-expressiong or control vectors followed by LPS stimulation. MiR-34b-5p was confirmed as a target of TUG1 using dual-luciferase reporter assay. GRB2 associated binding protein 1 (GAB1) was confirmed as a downstream target of miR-34b-5p using the same method. In the rescue experiment, PMVECs were co-transfected with TUG1-expressing vector and miR-34b-5p mimics (or control mimics) 24 h before LPS treatment.

Results: ALI mice showed reduced levels of TUG1, pulmonary injury, and induced apoptosis and inflammation compared to the control group. The overexpression of TUG1 in ALI mice ameliorated sepsis-induced pulmonary injury, apoptosis and inflammation. TUG1 also showed protective effect in LPS-treated PMVECs. The expression of MiR-34b-5p was negatively correlated with the level of TUG1. TUG1-supressed apoptosis and inflammation in LPS-stimulated PMVECs were restored by miR-34b-5p overexpression. GAB1 was inversely regulated by miR-34b-5p but was positively correlated with TUG1 expression.

Conclusion: TUG1 alleviated sepsis-induced inflammation and apoptosis via targeting miR-34b-5p and GAB1. These findings suggested that TUG1 might be served as a therapeutic potential for the treatment of sepsis-induced ALI.

Keywords: Acute lung injury; Apoptosis; GAB1; Inflammation; Sepsis; TUG1; miRNA.

Fig. 1

The level of TUG1 in the lung tissues of septic mice and the effect of TUG1 overexpression in sepsis-induced ALI. Adult C57BL/6 mice were randomly assigned into 4 groups: sham, CLP, CLP + Ad-GFP, and CLP + Ad-TUG1 (n = 12 per group). CLP, CLP + Ad-GFP, CLP + Ad-TUG1 groups received intravenous injections of vehicle, control adenoviral vector, and adenoviral vector expressing TUG1, respectively, followed by the CLP operation. Animals in the sham group underwent the same CLP procedure without the ligation or puncture of the cecum. (a) The expression of TUG1 in mouse lung tissues were examined using qRT-PCR. (b) Survival rate of mice within 72 h following CLP procedure. (c) Paraffin-embedded lung tissue samples were stained for H&E and the degree of lung damage was determined using lung injury scoring. Representative histological images were shown at 400× magnification. (d) The serum level of TUG1 in ARDS patients (n = 35) and healthy subjects (n = 68) was measured using a ELISA kit

Fig. 2

Impact of TUG1 upregulation on CLP-induced apoptosis and inflammation in mouse lung tissues. (a) Histological analysis of sectioned mouse lung tissue samples using TUNEL staining and immunohistochemistry for caspase-3 expression. Representative histological images (400× magnification) and the percentage of positively stained cells were shown. (b) The levels of Bax, Bcl-2, and cleaved PARP in the lung tissues of all mice were analyzed using Western blot. Representative plots (left) and bar charts (right) were shown. (c-d) The mRNA and proteins expressions of TNF-α, IL-1β and IL-6 in all animals were detected using qRT-PCR and ELISA, respectively. (e) The protein expressions of IL-4 and IL-10 in mouse lung tissues were measured using ELISA

Fig. 3

Overexpression of TUG1 mediated the levels of apoptotic markers and proinflammatory cytokines in LPS-treated PMVECs. PMVECs were divided into 4 groups: control (Ctrl), LPS, Vector, and TUG1. LPS, Vector, and TUG1 groups were cultured with serum-free medium containing nothing, control adenoviral vector, and adenoviral vector expressing TUG1, respectively, followed by the 6-h stimulation of 100 ng/mL LPS. Ctrl group remained untreated. (a) Relative expression of TUG1 were measured using qRT-PCR 6 h after LPS treatment. (b) TUNEL and DAPI staining were performed to detect apoptotic cell death in PMVECs. The number of TUNEL-positive cells were counted in six randomly selected fields per slide. (c) The expression of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP in all groups of PMVECs were examined using Western blot. Representative plots (left) and bar charts (right) were shown. (d-e) The mRNA and proteins levels of TNF-α, IL-1β and IL-6 in PMVECs were assessed using qRT-PCR and ELISA, respectively

Fig. 4

TUG1 targeted on miR-34b-5p in the models of sepsis. (a) The putative binding site between TUG1 and miR-34b-5p were predicted on miRDB. The wild-type binding sequence and the designed mutant sequence were indicated as TUG1-WT and TUG1-MUT, respectively. (b) PMVECs were transfected with miR mimics or miR-34b-5p mimics. The transfection efficacy was determined by measuring the level of miR-34b-5p in transfected cells. (c) HEK-293 T cells were co-transfected with miR mimics (or miR-34b-5p mimics) and TUG1-WT (or TUG1-MUT). The luciferase activity was measuring 48 h post transfection using dual-luciferase reporter assay. (d) The RIP assay was performed in PMVECs using a mouse monoclonal anti-Ago2 antibody as a positive control and an anti-IgG antibody as a negative control. The expressions of TUG1 and miR-34b-5p in RISCs were analyzed using qRT-PCR. (e) The relative expression of miR-34b-5p in PMVECs transfected with control adenoviral vector (Vector) or adenoviral vector expressing TUG1(TUG1) were examined using qRT-PCR. (f) The relative expression of miR-34b-5p in sham- and CLP-operated mice were detected using qRT-PCR. (g) The correlation between miR-34b-5p expression and TUG1 level in mouse lung tissues were analyzed using Spearman’s rank correlation test

Fig. 5

GAB1 is a target gene of miR-34b-5p. (a) GAB1 was predicted as a downstream target of miR-34b-5p using TargetScan. The putative GAB1 binding site for miR-34b-5p (GAB1-WT) and the designed mutant sequence (GAB1-MUT) were shown. (b) HEK-293 T cells co-transfected with miR mimics (or miR-34b-5p mimics) and GAB1-WT (or GAB1-MUT) were analyzed for luciferase activity 48 h after transfection using dual-luciferase reporter assay. (c) PMVECs were transfected with miR mimics or miR-34b-5p mimics. The expression of GAB1 in transfected PMVECs were examined using Western blot. (d) The relative expression of GAB1 in sham- and CLP-operated animals were assessed using qRT-PCR. (E) The correlation between miR-34b-5p level and GAB1 expression in mouse lung tissues were examined using Spearman’s rank correlation test. (F) The protein expression of GAB1 in mice was measured using Western blot

Fig. 6

TUG1 regulated sepsis-induced apoptosis and inflammation via the modulation of miR-34b-5p and GAB1. PMVECs were divided into 3 groups: Vector+miR mimics, TUG1 + miR mimics and TUG1 + miR-34b-5p mimics. Cells in Vector+miR mimics group were co-transfected with control adenoviral vector and control mimics. PMVECs in TUG1 + miR mimics and TUG1 + miR-34b-5p mimics groups were co-transfected with adenoviral vector expressing TUG1 and control mimics (or miR-34b-5p mimics). LPS stimulation was performed 24 h post transfection. (a) The protein levels of GAB1, Bax, Bcl-2, and cleaved caspas-3, and cleaved PARP in all groups were determined using Western blot. Representative plots (left) and bar charts (right) were shown. (b) The production of TNF-α, IL-1β and IL-6 in co-transfected PMVECs were measured using ELISA