去泛素化酶USP45在高氧诱导支气管肺发育不良疾病中的生理功能和调控机制研究

BPD is the most common chronic lung disease in infants and the long-term consequences extend well into adulthood. Hyperoxia-induced acute lung injury (HALI) is a key contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) in neonates, which is characterized by impaired alveolarization and dysregulated vascularization. Currently no specific preventive or therapeutic agent is available in treating BPD and the genetic evidences of multiple regulators contributing to the initiation or progression of BPD are rather weak. . Ubiquitination play pivotal roles in maintaining intracellular protein homeostasis and signaling. Deubiquitylase, the core component of the ubiquitination machinery, governs protein stability and activity by reversing ubiquitin from modified substrates. Current researches have demonstrated the importance of deubiquitylase in regulating multiple biological processes including cell differentiation, cell division, fate specification and migration, while the role of deubiquitylase in the pathogenesis of lung development was largely unveiled. In this study, We identified USP45, a family member of the largest subgroup of deubiquitylase, as a hyperoxia responsive element in the pathological process of HALI/BPD through a combination of integrative analysis for significantly differently expressed genes in GEO among BPD neonates and an unbiased cell-level screening of the USP families in response to hyperoxia stimulation,. . Of note, we discovered that hyperoxia induces USP45 expression in lung type II epithelial cells (T2AECs) of newborn mice. Meanwhile, the expression level of USP45 can be gradiently up-regulated in mouse lung epithelial cell MLE12 and human lung epithelial cell Beas-2B, in an oxygen concentration dependent manner. We further experimentally validate the mechanistic angiogenic, inflammatory, cell death and cell proliferation pathways of USP45 in vitro and observed that when USP45 was depleted in lung epithelial cells, cell proliferation, cell migration levels were notably increased, and apoptosis as well as EMT level was significantly attenuated. Furthermore, down-regulated USP45 inhibits the caspase3 cleavage and TGF-β/smads pathway whereas enhances Ang1/Tie2 signaling pathways. Based on the hypothesis that USP45 promotes apoptosis and impairs alveolarization of epithelial cells (AEC), we intended to conduct our systematic research in molecular, cellular, mouse levels. Initially, we will observe that whether global or T2AEC specific-USP45 knockout can ameliorate the phenotype of BPD in neonatal mice; subsequently, we will determine the influence of USP45 knockout on alveolarization formation, cell apoptosis, proliferation, migration, epithelial-mesenchymal transition, angiogenesis as well inflammatory infiltration of T2AEC in BPD. Moreover, USP45 stably depleted MLE12 and primary USP44 KO T2AEC will be utilized to discover the underlying mechanisms of USP45 regulated targets by IP-MS metho, which will be further confirmed in the mouse BPD model. Collectively, these studies will be of great help in unraveling the role of USP45 in regulating BPD and thus provide novel insights into prevention and treatment of the disease.

支气管肺发育不良是新生儿最常见的慢性疾病并伴随终身。高氧诱导的急性肺损伤是该病的主要诱因之一，其典型病理特征为肺泡形成受阻和血管生成失调，目前临床上尚无特异性干预手段且已报道的调节因子遗传学证据不足。去泛素化酶作为蛋白质活性及含量的重要调节者被报道参与众多生理学过程，但其在肺发育和病理过程中的调控报道甚少。本研究从BPD临床样本的GEO数据分析入手，筛选到显著性差异表达基因去泛素化酶USP45。在高氧诱导的细胞和小鼠模型中，USP45表达水平显著上调；敲低USP45抑制肺上皮细胞凋亡、EMT，促进增殖和迁移；活化caspase3切割、增强Ang1/Tie2通路，抑制TGF-β/Smads通路。本项目拟在此基础上，从分子机制、细胞效应、小鼠整体表型等多个角度揭示USP45在小鼠支气管肺发育不良中的调控靶标和作用机制，为阐明人类该病的发病机制和临床救治提供新思路。

支气管肺发育不良是新生儿最常见的慢性疾病并伴随终身。高氧诱导的急性肺损伤是该病的主要诱因之一，其典型病理特征为肺泡形成受阻和血管生成失调，目前临床上尚无特异性干预手段且已报道的调节因子遗传学证据不足。去泛素化酶作为蛋白质活性及含量的重要调节者被报道参与众多生理学过程，但其在肺发育和病理过程中的调控报道甚少。本研究从BPD临床 样本的GEO数据分析入手，筛选到显著性差异表达基因去泛素化酶USP45。在高氧诱导的细胞和 小鼠模型中，USP45表达水平显著上调；敲低USP45抑制肺上皮细胞凋亡、EMT，促进增殖和迁 移；活化caspase3切割、增强Ang1/Tie2通路，抑制TGF-β/Smads通路。本项目从分子机制、细胞效应、小鼠整体表型等多个角度揭示USP45-Ang1信号轴在小鼠支气管肺发育不良中的调控靶标和作用机制，为阐明人类该病的发病机制和临床救治提供新思路。