中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (11): 1692-1700.doi: 10.12307/2023.155

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

高氧诱导miRNA差异性表达介导新生大鼠的肺损伤

王丽娜，章  容，康  兰，雷小平，何红霞，董文斌   

  1. 西南医科大学附属医院新生儿科，四川省出生缺陷中心，四川省泸州市  646000
  • 收稿日期:2022-03-15 接受日期:2022-05-21 出版日期:2023-04-18 发布日期:2022-09-27
  • 通讯作者: 董文斌，博士生导师，教授，主任医师，西南医科大学附属医院新生儿科，四川省出生缺陷中心，四川省泸州市 646000
  • 作者简介:王丽娜，女，1993年生，重庆市人，汉族，西南医科大学医学硕士，初级医师，主要从事新生儿支气管肺发育不良机制研究。
  • 基金资助:
    国家自然科学基金资助项目(81571480)，项目名称：去乙酰化酶SIRT1的SDMO修饰与早产儿高氧肺损伤的临床实验分析，项目负责人：董文斌

Hyperoxia-induced differential expression of microRNAs mediates lung injury in neonatal rats

Wang Lina, Zhang Rong, Kang Lan, Lei Xiaoping, He Hongxia, Dong Wenbin   

  1. Department of Neonatology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2022-03-15 Accepted:2022-05-21 Online:2023-04-18 Published:2022-09-27
  • Contact: Dong Wenbin, Doctoral supervisor, Professor, Chief physician, Department of Neonatology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Wang Lina, Master, Physician, Department of Neonatology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81571480 (to DWB)

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摘要：

文题释义：
支气管肺发育不良：属于慢性肺部疾病，是围产医学研究的重点疾病之一，多发生于早产儿，但目前没有有效的治疗方式。新“支气管肺发育不良”的主要病理特征是肺发育停滞和轻微的肺损伤，早产使胎儿肺提前结束宫内发育过程，与肺发育停滞似乎存在必然联系，而生后需要持续的氧疗，会造成不成熟肺组织的二次伤害。因此，早产和高氧必然会诱发机体内分子表达的改变，最终导致支气管肺发育不良的发生，其发病的分子机制则是此次研究的主要目的。
差异表达miRNA：miRNA是转录组的保守基因序列，能够调控靶蛋白的抑制或降解，在个体的生长、发育和疾病中起着关键作用，且能够调控肺发育整个时期(胚胎期、假腺期、小管期、囊状期、肺泡期)。又由于早产儿的肺发育在囊状期之前的存活率极低。以此为基础，该研究旨在寻找高氧暴露后随时间动态变化的miRNA及其下游蛋白，探索与支气管肺发育不良发病机制相关的囊状期和肺泡期的信号通路，揭示支气管肺发育不良发病中的转录调控机制。

背景：支气管肺发育不良主要的病理特征是肺发育停滞和较轻微的组织结构损伤，胎儿肺发育需经历5个时期，到囊状期的胎儿生后才可能存活，因此，早产、高氧诱导支气管肺发育不良的发病机制研究重点关注了囊状期和肺泡期。
目的：探讨2个发育阶段——囊状期和肺泡期中起调控作用的差异miRNA在高氧相关的支气管肺发育不良中的作用。
方法：将新生SD大鼠随机分为4组：0 d组(n=10)出生后即取肺组织；4，7，14 d组均设置2个亚组，高氧组(n=10)出生后进入持续高氧环境中(氧浓度85%-90%)，分别于4，7，14 d后取肺组织，空气组(n=10)出生后进入空气环境中(吸入氧浓度21%)，分别于4，7，14 d后取肺组织。采用miRNA-Seq技术获取肺组织miRNA并建库，Illumina平台测序，Deseq2(Version 1.10.1)、Venn图组间比较依次筛选DEmiRs，GO富集、PPI分析预测差异表达的miRNAs靶蛋白。
结果与结论：①0 d组有633个miRNA，4，7，14 d组中的高氧亚组分别有609，614，584个miRNA，空气亚组分别有629，608，581个miRNA；②在出生0-4 d(囊状期)、4-7 d、4-14 d(肺泡期)时，高氧组分别获得130，3，114个差异表达的miRNAs，空气组分别获得106，0，111个差异表达的miRNAs，各时期两组均差异表达的miRNAs分别为91，0，79个；③在囊状期和肺泡期，高氧组新生鼠肺组织中miR-34a-5p、miR-21-5p显著表达，miR-34a-5p靶蛋白有198个，围绕Pdgfrb/Pdgfra/Ngfr；miR-21-5p靶蛋白302个，围绕Mapk1/Stat3；④结果显示，miR-34a-5p、miR-21-5p各自在囊状期和肺泡期受高氧诱导显著表达，分别调控靶蛋白PDGFRB/PDGFRA/NGFR、MAPK1/Stat3，介导阶段性肺发育过程的信号通路，参与支气管肺发育不良的发生发展。

https://orcid.org/0000-0003-2780-807X(王丽娜)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 支气管发育不良, miRNA高通量测序, miRNA-21-5p, miR-34a-5p, MAPK1/Stat3, PDGFRB/PDGFRA/NGFR

Abstract: BACKGROUND: The main pathological features of bronchopulmonary dysplasia are stagnation of lung development and minor tissue damage. Fetal lung development needs to go through five stages, and a preterm infant cannot survive after birth until the cystic stage. Therefore, research on the pathogenesis of premature birth- and hyperoxia-induced bronchopulmonary dysplasia has focused on cystic and alveolar phases.
OBJECTIVE: To investigate the regulatory role of differentially expressed microRNAs in hyperoxia-associated bronchopulmonary dysplasia during two developmental stages – cystic and alveolar stages.
METHODS: Newborn Sprague-Dawley rats were randomly into four groups: in 0 day group (n=10), rat lung tissue was taken immediately after birth; 4-, 7-, 14-day groups were further divided into two subgroups – hyperoxia group (exposed to persistent hyperoxia with an oxygen concentration of 85%-90%, n=10) and air group (exposed to the air with FiO2: 21%, n=10). Lung samples tissue were then taken at postnatal days 4, 7, 14. MicroRNAs in lung tissue were acquired using miRNA-Seq technology to build a library, and sequenced on the Illumina platform. Differently expressed microRNAs between different groups were screened by Deseq2 (Version 1.10.1) and Venn map. Gene ontology enrichment and protein-protein interaction analysis were performed to predict target proteins of differentially expressed microRNAs. 
RESULTS AND CONCLUSION: There were 633 microRNAs in the 0-day group, 609, 614, and 584 microRNAs in the hyperoxia group at postnatal days 4, 7, 14, respectively, and 629, 608, and 581 in the corresponding air groups. The hyperoxia group obtained 130, 3, and 114 differentially expressed microRNAs during postnatal days 0-4 (cystic stage), 4-7, and 4-14 (alveolar stage), respectively. And the corresponding air group received 106, 0, 111 differentially expressed microRNAs respectively. There were 91, 0, and 79 differentially expressed microRNAs in the two groups at different stages. During the cystic and alveolar phases, miR-34a-5p and miR-21-5p were significantly expressed in the lung tissue of neonatal rats in the hyperoxia group. miR-34a-5p had 198 target proteins, which concentrated around the Pdgfrb/Pdgfra/Ngfr; miR-21-5p had 302 target proteins that mainly revolved around the Mapk1/Stat3. To conclude, miR-34a-5p and miR-21-5p are highly expressed in the cystic and alveolar stages under hyperoxia exposure, targeting the proteins about PDGFRB/PDGFRA/NGFR and MAPK1/Stat3 respectively, which mediate the process of lung development and participate in the occurrence and development of bronchopulmonary dysplasia. 

Key words: bronchopulmonary dysplasia, microRNA high-throughput sequencing, miRNA-21-5p, miR-34a-5p, MAPK1/Start3, PDGFRB/PDGFRA/NGFR

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