TET2巴豆酰化介导BCL2基因高甲基化在COPD肺血管内皮细胞凋亡和功能障碍中的作用及机制研究

Recent studies confirmed that cigarette smoke caused abnormal hypermethylation of genes played a noval role in pulmonary vascular endothelial cells (PVECs) apoptosis and dysfunction of COPD. DNA dioxygenase Ten-Eleven-Translocation protein 2 (TET2) deletion is the initiating factor of gene hypermethylation, which can reduce the recruitment of the classic transcription factor PU.1, leading to gene inactivation. Lysine crotonylation (Kcr) is a brand new and promising modification to regulate protein expression and activity. We observed that TET2 protein was downregulated in COPD lung tissue, while Kcr levels of TET2 increased. We also found the expression of TET2 associated with the anti-apoptotic gene BCL2 promoter hypermethylation and endothelial apoptosis. Preliminary studies suggested BCL2 promoter hypermethylation that triggered by smoking, caused mitochondrial cytC releasing and led to apoptosis and dysfunction happened in PVECs. Therefore, we hypothesis that smoking-induced crotonylation of TET2 might cause BCL2 promoter hypermethylation and decrease the binding of PU.1, eventually it could initiate the apoptosis and dysfunction of PVECs. Our research will investigate the role of TET2 in regulating PVECs apoptosis of COPD induced by BCL2 promoter methylation. Furthermore, we will elucidate the detailed mechanism of TET2 crotonylation through constructing TET2 conditional knockout mice and lysine sites targeted mutation. Execution of these studies may provide a new epigenetic option in the prevention and treatment of COPD.

近年研究证实吸烟所致基因异常高甲基化调控肺血管内皮细胞凋亡和功能障碍在COPD发病中起重要作用。DNA双加氧酶TET2缺失是基因高甲基化的始动因素，可减少转录因子PU.1募集抑制基因转录。赖氨酸巴豆酰化（Kcr）是新近发现的调控蛋白表达和活性的关键修饰方式。我们观察到COPD肺组织TET2蛋白表达下调而Kcr水平增高，且与抗凋亡基因BCL2启动子区高甲基化和内皮功能受损相关；吸烟触发BCL2基因高甲基化引起线粒体cytC释放致肺内皮细胞凋亡。故我们推测吸烟诱导TET2高巴豆酰化引起BCL2基因高甲基化，减少BCL2启动子区PU.1的结合，启动肺内皮细胞凋亡和功能障碍参与COPD的形成。申请者拟通过构建TET2条件性敲除鼠和定向诱变赖氨酸位点，探讨TET2调控BCL2基因甲基化在COPD肺内皮细胞凋亡中的作用及其巴豆酰化的分子生物学机制，为COPD表观遗传学防治提供全新的干预途径及理论依据。

表观遗传通过弥合遗传变异与环境危险因素调控COPD（Chronic obstructive pulmonary disease, COPD）的发生发展。基因关联研究显示DNA去甲基化酶TET2（Ten-Eleven-Translocation protein 2, TET2）的遗传变异与低FEV1（Forced expiratory volume in one second, FEV1）显著相关，吸烟是COPD的环境危险因素，气道上皮细胞凋亡是COPD的发病机制之一，然而TET2参与COPD发病的具体机制尚未明了。TET2活性可受泛素化修饰调控，USP21（ubiquitin-specific peptidase 21, USP21）是去泛素化酶，但是目前尚未有研究探索USP21与TET2蛋白活性的关联。本研究旨在探究TET2蛋白在CSE诱导的气道上皮细胞凋亡中的作用机制并探讨USP21与TET2蛋白活性的关系。本课题通过慢性阻塞性肺疾病（COPD）病人、动物模型和细胞三个层面对TET2在COPD中的作用及其调控机制进行了研究。我们的研究显示在COPD患者中TET2表达下调并伴随抗凋亡基因BCL2的表达下调。本次实验采用烟熏6月的方法成功建立了COPD小鼠模型。在动物实验中我们同样发现COPD小鼠模型肺组织TET2的蛋白表达明显下调并伴随抗凋亡基因BCL2水平下调，而进一步采用TET2全身敲除鼠构建COPD小鼠模型则发现，TET2敲除会进一步加剧烟熏导致的慢阻肺发生。采用CSE处理TET2过表达稳转株，或者我们同样发现TET2缺失介导CSE引起的凋亡和氧化应激过程。我们研究发现TET2蛋白通过泛素蛋白酶体途径降解；通过过表达质粒筛选，我们发现去泛素化酶USP21特异性介导TET2蛋白的降解。荧光定位及免疫共沉淀显示USP21和TET2存在相互作用。沉默USP21可加速PRMT6的降解，进而影响DNA甲基化并抑制抗凋亡基因转录，CSE可抑制USP21的表达，而过表达USP21则可部分挽救TET2的降解。同时本项目还拓展了课题相关的基础研究包括PU.1能够调控香烟烟雾所致内皮祖细胞肺部归巢和生物学功能障碍、二氢槲皮素通过Nrf2相关通路抑制慢阻肺发病过程中香烟烟雾所诱导的铁死亡以及TET2能够通过靶向GPX4调节香烟烟雾诱导的支气管上皮细胞铁死亡等新机制。

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