miR-21-5p经PI3K/Akt通路在HALI调控AECⅡ凋亡的研究

Hyperoxic acute lung injury (HALI) is the most typical complication happens in oxygen therapy, and may develop acute adult respiratory distress syndrome and neonatal bronchopulmonary dysplasia as well, which is also one of the the main causes of death in patients and neonatal disability. Type Ⅱ alveolar epithelial cells (AECⅡ) apoptosis is the pivotal step in hyperxia toxicity. Thus, the inhibition of this process can achieve treatment of HALI. But there are few intervention found works. Our previous study found that miR-21-5p significantly antagonized AECⅡ apoptosis. We presume that miR-21-5p may connected with negative regulating PTEN/PDCD4 mRNA expression, which are upstream and downstream molecules in PI3K/Akt pathway respectively, through analysis of miRNA targeting genes predict software(TarBase and miRTarBase). which are upstream and downstream molecules in PI3K/Akt pathway, This presumption worth to study further to testify its accuracy. Our study aims at validating if the PTEN/PDCD4 are miR-21-5p target genes,and,approach whether miR-21-5p was involved in PI3K/Akt pathway to protect AECⅡfrom apoptosis and thus cure the HALI with the presence of PI3K inhibitor LY294002 through vivo/vitro way. In summary, this study will provide the theoretical basis for gene therapy HALI, and suggesting new targets for small molecule drug intervention.

高氧性急性肺损伤(HALI)是氧疗最典型的并发症，可发展为成人急性呼吸窘迫综合征及新生儿支气管肺发育不良，是导致患者死亡及新生儿残疾的主要原因之一。Ⅱ型肺泡上皮细胞（AECⅡ）凋亡是高氧毒性的核心环节，抑制该过程可有效治疗HALI，但目前尚缺乏干预AECⅡ凋亡的有效措施。本课题组前期研究发现miR-21-5p可有效拮抗AECⅡ凋亡，经miRNA靶基因预测软件分析，推测其抑制AECⅡ凋亡机制可能与负向调控PI3K/Akt通路的上下游分子PTEN和PDCD4的表达有关。这一推论是否正确，值得深入研究。故本研究拟验证PTEN和PDCD4 是否为miR-21-5p的靶基因，并结合PI3K抑制剂LY294002的采用，探讨体内外miR-21-5p是否通过调控PI3K/Akt通路保护AECⅡ免受凋亡，并对HALI具有治疗作用。综上，本研究将为HALI的基因治疗提供理论依据，提示新的小分子药物干预靶点。

氧疗被广泛应用于呼吸衰竭等严重低氧血症患者的治疗，对防止器官功能衰竭、挽救患者生命具有重要意义，然而高氧性急性肺损伤（HALI）仍然是长期持续吸入高浓度氧的主要并发症之一。目前研究发现高氧会引起肺泡上皮细胞(AEC)的凋亡和坏死，细胞凋亡是调节高氧毒性的核心环节。肺泡II型上皮细胞(AECⅡ)是 AEC的干细胞，也是高氧攻击的主要细胞，抑制其凋亡可有效减轻实验大鼠HALI 程度。因此，AEC Ⅱ凋亡调控很可能成为临床防治 HALI 的有效措施。近年研究发现，microRNA(miRNA)作为表观遗传修饰的调控靶点，通过三种形式负性调控这人体约60%的基因表达，其中miRNA-21是最受关注的miRNAs分子之一。本课题组前期通过基因芯片技术研究表明miR-21-5p在高氧损伤后低表达，可能是AECII内重要的抗凋亡基因。然后运用生信分析、应用实时定量PCR、Western blot 技术及双荧光素酶报告基因法验证miR-21-5p可靶向PTEN、PDCD4 基因激活PI3K/AKT信号通路发挥其负向调控分子的功能。然后通过在肺泡II型上皮细胞中过表达miR-21-5p 后抑制PI3K/AKT通路；进行动物实验，建立HALI 模型；过表达miRNA-21-5p后特异性阻断PI3K/Akt 通路， 结合运用遗传学、蛋白质生物化学、细胞生物学、流式细胞术等方法与技术，结果表明miRNA-21-5p抑制AECⅡ凋亡的机制之一是通过靶向PTEN及PDCD4激活PI3K/AKT信号通路调控抗凋亡蛋白的表达来实现的。此研究为进一步阐明高氧性肺损伤机制、寻找更好的肺保护措施提供理论依据。