Role of signaling pathway of long non-coding RNA growth arrest-specific transcript 5/microRNA-200c-3p/angiotensin converting enzyme 2 in the apoptosis of human lung epithelial cell A549 in acute respiratory distress syndrome

Abstract

Objective: To investigate whether long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5)/microRNA-200c-3p/angiotensin converting enzyme 2(ACE2) involved in the regulation of the apoptosis of human lung epithelial cell A549 in acute respiratory distress syndrome (ARDS). Methods: ARDS rat models were established and were divided into control, ARDS, ARDS+ pcDNA and ARDS+ pcDNA-GAS5 groups. Six hours after the establishment of ARDS rat model, arterial blood and lung tissues of the rats from the four groups were collected. The changes of partial pressure of oxygen (PO(2)) and partial pressure of CO(2) (PCO(2)) were analyzed and the expression of GAS5 in lung tissue was observed in these groups. Then, A549 cells were divided into control, lipopolysaccharide (LPS), LPS+ pcDNA, LPS+ pcDNA-GAS5, LPS+ pcDNA-GAS5+ pre-NC, LPS+ pcDNA-GAS5+ miR-200c-3p mimic groups. Quantitative real-time PCR (qRT-PCR) was conducted to measure lncRNA GAS5, ACE2 and miR-200c-3p levels. RNA immunoprecipitation and RNA pull-down assay were used to detect the combination between GAS5 and miR-200c-3p. Western blotting was used to detect the protein level of ACE2. Flow cytometry was used to observe the apoptosis of A549 cells in those groups. The data between groups were compared by t test. Results: In ARDS rat model, PO(2) value was significantly increased in ARDS+ pcDNA-GAS5 group than that in ARDS+ pcDNA group[(81.5±3.3) vs (57.5±5.1) mmHg, t=4.850, P<0.05], and PCO(2) value was significantly decreased in ARDS+ pcDNA-GAS5 group than that in ARDS+ pcDNA group[(50.6±1.9) vs (64.0±1.9) mmHg, t=5.940, P<0.05]. LncRNA GAS5 level in A549 cells of LPS group decreased significantly than that in control group (0.43±0.01 vs 1.01±0.01, t=0.242, P<0.05). Compared with LPS+ pcDNA group, ACE2 expression increased significantly in LPS+ pcDNA-GAS5 group (0.85±0.04 vs 0.34±0.02, t=1.800, P<0.05). Compared with LPS+ pcDNA-GAS5+ pre-NC group, ACE2 expression decreased significantly in LPS+ pcDNA-GAS5+ miR-200c-3p mimic group (0.62±0.01 vs 0.84±0.02, t=9.440, P<0.05). Compared with control group, the percentage of A549 cell apoptosis promoted significantly in LPS group (25.90±0.61 vs 7.90±0.22, t=0.257, P<0.05). Compared with LPS+ pcDNA group, the percentage of A549 cell apoptosis suppressed significantly in LPS+ pcDNA-GAS5 group (10.50±0.37 vs 26.37±0.45, t=1.760, P<0.05). Compared with LPS+ pcDNA-GAS5+ pre-NC group, the percentage of A549 apoptosis promoted significantly in LPS+ pcDNA-GAS5+ miR-200c-3p mimic group (19.07±0.56 vs 10.87±0.26, t=0.643, P<0.05). Conclusion: In ARDS, down-regulation of lncRNA GAS5 decreases ACE2 expression through increasing miR-200c-3p to promote the apoptosis of A549 cells, thus to promote the progression of ARDS.