Effect of lncRNA-AC013472.3 on LPS-stimulated secretion of tumor necrosis factor-α in NR8383 rat alveolar macrophages

Abstract

Objective: To investigate the effect of long non-coding RNA-AC013472.3 on lipopolysaccharide (LPS)-stimulated secretion of tumor necrosis factor (TNF)-α in NR8383 rat alveolar macrophages. Methods: Silencing and overexpression models of lncRNA-AC013472.3 were established with NR8383 rat alveolar macrophages as the experimental subjects. The silencing models were divided into three groups: random nonsense negative small interfering RNA sequence (si-con) group (si-con group, si-con transfected NR8383 cells), LPS+si-con group (10 μg/L LPS was used to treat si-con transfected NR8383 cells for 24 h), and siRNA group (siRNA transfected NR8383 cells), and LPS+siRNA group (10 μg/L LPS was used to treat siRNA transfected NR8383 cells for 24 h). The overexpression models were divided into the empty plasmid (p-con) group (p-con transfected NR8383 cells), LPS+p-con group (10 μg/L LPS was used to treat p-con transfected NR8383 cells for 24 h), lncRNA overexpression plasmid (plncRNA) group (plncRNA transfected NR8383 cells), and the LPS+plncRNA group (10 μg/L LPS was used to treat plncRNA transfected NR8383 cells for 24 h). The mRNA levels of TNF-α in each group were examined by quantitative real-time PCR (qPCR). The protein levels of tumor necrosis factor receptor-related factor-6 (TRAF-6) and phosphorylated nuclear factor-κB (NF-κB) p65 were examined by Western blot. Results: In the silencing model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+si-con group were significantly higher than those in the si-con group (2.040±0.195 vs 1.048±0.207, 0.473±0.022 vs 0.293±0.076 and 0.469±0.062 vs 0.252±0.038)(all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+siRNA group were significantly higher than those in the siRNA group (4.158±0.119 vs 1.028±0.019, 0.700±0.104 vs 0.231±0.023 and 0.771±0.095 vs 0.258±0.050)(all P<0.05). The relative expression levels of all indexes in the LPS+siRNA group were significantly higher than those in the LPS+si-con group (all P<0.05). In the overexpression model, the mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+p-con group were significantly higher than those in the p-con group (1.961±0.169 vs 0.999±0.143, 0.533±0.047 vs 0.247±0.020 and 0.565±0.108 vs 0.276±0.048) (all P<0.05). The mRNA levels of TNF-α, the protein levels of TRAF-6 and NF-κB p65 in the LPS+plncRNA group were significantly higher than those in the plncRNA group (1.322±0.110 vs 1.043±0.093, 0.347±0.035 vs 0.232±0.023 and 0.405±0.072 vs 0.268±0.031) (all P<0.05). The relative expression of all indexes in the LPS+plncRNA group were significantly lower than that in the LPS+p-con group (all P<0.05). Conclusion: LncRNA-AC013472.3 may inhibit the activation of NF-κB signaling pathway, thereby inhibiting the LPS-stimulated secretion of TNF-α in NR8383 rat alveolar macrophages.