长链非编码RNA生长阻滞特异转录物5／微小RNA 200c-3p／血管紧张素转换酶2信号轴参与呼吸窘迫综合征人肺上皮细胞A549的凋亡  

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

作　　者：李宏宾[1] 訾盼盼[1] 石慧娟[1] 高敏[1] 孙荣青[1] Li Hongbin;Zi Panpan;Shi Huijuan;Cao Min;Sun Rongqing(Department of Critical Care Medicine,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院重症医学科,郑州450052

出　　处：《中华医学杂志》2018年第41期3354-3359,共6页National Medical Journal of China

摘　　要：目的探究长链非编码RNA生长阻滞特异转录物5／微小RNA200e-3p／血管紧张素转换酶2（1ncRNAGAS5／miR-200c-3p／ACE2）信号轴是否参与呼吸窘迫综合征（ARDS）肺上皮细胞的凋亡。方法构建ARDS大鼠模型，实验分为对照组（Control组）、LPS处理组（ARDS组）、LPS＋pcDNA处理组（ARDS＋pcDNA组）和LPS＋pcDNA-GAS5处理组（ARDS＋pcDNA-GAS5组）。ARDS大鼠构建6h后收集4组大鼠动脉血及肺组织，血气分析仪进行测定氧分压（PaO2）和二氧化碳分压（PaCO2），并观察GAS5的表达变化。随后，用LPS处理人肺泡Ⅱ型上皮细胞A549，分为未处理、LPS、LPS＋pcDNA、LPS＋pcDNA-GAS5、LPS＋pcDNA-GAS5＋pre-NC和LPS＋pcDNA-GAS5＋miR-200c．3pmimic组。实时荧光定量聚合酶链反应（qRT-PCR）检测lncRNAGAS5、ACE2和miR-200c-3p的表达水平；RNA免疫沉淀和RNA下拉（pull-down）实验用于检测GAS5与miR．200c-3p的结合与调控：Westernblotting法检测ACE2的蛋白表达；流式细胞术用于检测A549细胞的凋亡。组间数据比较采用t检验。结果ARDS大鼠实验表明与ARDS＋pcDNA组相比，ARDS＋pcDNA-GAS5组P0，值显著升高[（81．5±3．3）比（57．5±5．1）mmHg，t=4．850，P〈0．05]，PC02值显著降低[（50．6±1．9）比（64．0±1．9）mmHg，t=5．940，P〈0．05]。细胞实验中，与Control组相比，LPS组A549细胞中IncRNAGAS5的表达显著下降（0．43±0．01比1．01±0．01，t=0．242，P〈0．05）。与LPS＋pcDNA组相比，LPS＋pcDNA-GAS5组ACE2的表达水平显著升高（0．85±0．04比0．34±0．02，t=1．800，P〈0．05）：与LPS＋pcDNA-GAS5＋pre-NC组相比，LPS＋pcDNA-GAS5＋miR-200c-3pmimic组ACE2的表达水平显著降低（0．62±0．01比0．84±0．02，t=9．440，P〈0．05）。与未处理组相比，LPS组A549细胞凋亡百分比明显升高（25．90±0．61比7．90±0．22，t=0．257，P〈0．05）；与LPS＋pcDNA组相比，LPS＋pcDNA-GAS5组凋亡百分比明显降低�Objective To investigate whether long non-coding RNA （lncRNA） growth arrestspecific 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 （ PO2 ） and partial pressure of CO2 （ PCO2 ） 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 ceils in those groups. The data between groups were compared by t test. Results In ARDS rat model, PO2 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 PCO2 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�

关 键 词：呼吸窘迫综合征 生长阻滞特异转录物5 微小RNA-200c-3p 血管紧张素转换酶2 人肺上皮细胞A549 

分 类 号：R563.8[医药卫生—呼吸系统]