Li H B, Zi P P, Shi H J, Gao M, Sun R Q
Department of Critical Care Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Yi Xue Za Zhi. 2018 Nov 6;98(41):3354-3359. doi: 10.3760/cma.j.issn.0376-2491.2018.41.013.
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). 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 test. 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, =4.850, <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, =5.940, <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, =0.242, <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, =1.800, <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, =9.440, <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, =0.257, <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, =1.760, <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, =0.643, <0.05). 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.
探讨长链非编码RNA（lncRNA）生长停滞特异性转录本5（GAS5）/微小RNA-200c-3p/血管紧张素转换酶2（ACE2）是否参与急性呼吸窘迫综合征（ARDS）中人类肺上皮细胞A549凋亡的调控。建立ARDS大鼠模型，并将其分为对照组、ARDS组、ARDS+pcDNA组和ARDS+pcDNA-GAS5组。在ARDS大鼠模型建立6小时后，收集四组大鼠的动脉血和肺组织。分析氧分压（PO₂）和二氧化碳分压（PCO₂）的变化，并观察这些组肺组织中GAS5的表达。然后，将A549细胞分为对照组、脂多糖（LPS）组、LPS+pcDNA组、LPS+pcDNA-GAS5组、LPS+pcDNA-GAS5+pre-NC组、LPS+pcDNA-GAS5+miR-200c-3p模拟物组。采用定量实时聚合酶链反应（qRT-PCR）检测lncRNA GAS5、ACE2和miR-200c-3p水平。采用RNA免疫沉淀和RNA下拉实验检测GAS5与miR-200c-3p之间的结合。采用蛋白质免疫印迹法检测ACE2蛋白水平。采用流式细胞术观察这些组中A549细胞的凋亡情况。组间数据采用检验进行比较。在ARDS大鼠模型中，ARDS+pcDNA-GAS5组的PO₂值显著高于ARDS+pcDNA组[（81.5±3.3）vs（57.5±5.1）mmHg，=4.850，<0.05]，且ARDS+pcDNA-GAS5组的PCO₂值显著低于ARDS+pcDNA组[（50.6±1.9）vs（64.0±1.9）mmHg，=5.940，<0.05]。LPS组A549细胞中lncRNA GAS5水平显著低于对照组（0.43±0.01 vs 1.01±0.01，=0.242，<0.05）。与LPS+pcDNA组相比，LPS+pcDNA-GAS5组ACE2表达显著增加（0.85±0.04 vs 0.34±0.02，=1.800，<0.05）。与LPS+pcDNA-GAS5+pre-NC组相比，LPS+pcDNA-GAS5+miR-200c-3p模拟物组ACE2表达显著降低（0.62±0.01 vs 0.84±0.02，=9.440，<0.05）。与对照组相比，LPS组A549细胞凋亡百分比显著升高（25.90±0.61 vs 7.90±0.22，=0.257，<0.05）。与LPS+pcDNA组相比，LPS+pcDNA-GAS5组A549细胞凋亡百分比显著降低（10.50±0.37 vs 26.37±0.45，=1.760，<0.05）。与LPS+pcDNA-GAS5+pre-NC组相比，LPS+pcDNA-GAS5+miR-200c-3p模拟物组A549细胞凋亡百分比显著升高（19.07±0.56 vs 10.87±0.26，=0.643，<0.05）。在ARDS中，lncRNA GAS5的下调通过增加miR-200c-3p降低ACE2表达，从而促进A549细胞凋亡，进而促进ARDS的进展。
