Depression of long non-coding RNA SOX2 overlapping transcript attenuates lipopolysaccharide-induced injury in bronchial epithelial cells via miR-455-3p/phosphatase and tensin homolog axis and phosphatidylinositol 3-kinase/protein kinase B pathway

Abstract

Airway inflammation is associated with various respiratory diseases, and previous research has confirmed that long non-coding RNAs (lncRNAs) play imperative roles in inflammatory responses. However, the function of lncRNA SOX2 overlapping transcript (SOX2-OT) in airway inflammation remains enigmatic. This study aimed to investigate the effects of SOX2-OT on lipopolysaccharide (LPS)-induced cell injury in human bronchial epithelial cells, BEAS-2B, and its potential mechanisms. The results showed increased cell apoptotic ratio, production of inflammatory cytokines, higher expression of adhesion molecules and activation of NF-κB in LPS-stimulated BEAS-2B cells. In LPS-stimulated BEAS-2B cells, SOX2-OT up-regulation and miR-455-3p down-regulation emerged simultaneously. SOX2-OT knockdown or miR-455-3p over-expression restrained LPS-induced inflammation and injury. SOX2-OT sponged to miR-455-3p and functioned as a ceRNA. In addition, phosphatase and tensin homolog (PTEN) served as an endogenous target of miR-455-3p to modulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway and disturb the alleviated consequence of miR-455-3p over-expression on LPS-induced BEAS-2B cell inflammation and cell injury. Our data demonstrated that SOX2-OT plays a pivotal role in LPS-induced inflammation and injury in BEAS-2B cells and exerts its function through the miR-455-3p/PTEN axis and modulation of the PI3K/AKT pathway.

Keywords: PTEN-PI3K/AKT pathway; SOX2-OT; human bronchial epithelial cells; inflammation cell apoptosis; miR-455-3p.

Graphical abstract

Figure 1.

SOX2-OT knockdown suppressed inflammation and injury in LPS-treated BEAS-2B cells. (a-c) Cell viability (a), the production of inflammatory cytokines (b) and the expression of SOX2-OT (c) in BEAS-2B cells exposed to LPS at various concentrations; mean ± SD, n = 3, *P < 0.05, **P < 0.01 vs 0 μg/ml group. (d) Transfection efficiency of ASO-SOX2OT was confirmed by qRT-PCR; mean ± SD, n = 3, **P < 0.01. (e-h) Inflammatory cytokines levels (e), LDH activity (f), cellular apoptosis proportion (g), and protein expression levels (h) in 1 μg/ml LPS incubated BEAS-2B cells transfected with/without ASO-SOX2OT; mean ± SD, n = 3, **P < 0.01 vs Control group, ^#P < 0.01 vs LPS+ASO-NC group.

Figure 2.

The sponging relationship of SOX2-OT and miR-455-3p in BEAS-2B cells. (a) Putative binding site of miR-455-3p on SOX2-OT; (b) Dual-luciferase activity of BEAS-2B cells cotransfected with miR-455-3p mimic and SOX2OT-WT/SOX2OT-MUT luciferase reporters; (c) The expression of miR-455-3p after SOX2-OT knockdown in LPS-treated BEAS-2B cells; (d) RIP assay assured that SOX2-OT interacted with miR-455-3p in BEAS-2B cells. mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Figure 3.

MiR-455-3p directly targeted PTEN in BEAS-2B cells. (a) Putative binding site of PTEN on miR-455-3p; (b) Dual-luciferase activity in BEAS-2B cells cotransfected with miR-455-3p mimic and PTEN-WT/PTEN-MUT luciferase reporters; (c) Detection of PTEN mRNA expression after transfection of miR-455-3p mimic in LPS-treated BEAS-2B cells; (d) Detection of PTEN protein expression after transfection of miR-455-3p mimic in LPS-incubated BEAS-2B cells. mean ± SD, n = 3, *P < 0.05, **P < 0.01.

Figure 4.

Knockdown of SOX2-OT restrained PTEN expression by enhancing miR-455-3p in LPS-incubated BEAS-2B cells. (a-b) The mRNA (a) and protein (b) level of PTEN expression in BEAS-2B cells transfected with different vectors; (c) TNF-α, IL-1β and IL-6 levels in each group; (d) LDH activity in each group; (e) Cellular apoptosis proportion in each group; (f) Proteins expressions in each group; mean ± SD, n = 3, *P < 0.05 vs LPS+ASO-SOX2OT group.