轻度低氧刺激对脑微血管内皮细胞lncRNA-NOS3AS/eNOS转录及转录后调控的机制研究

High-altitude anoxia decompensation may cause cerebral dysfunction, and hypoxia signal transduction of endothelium-derived NO mediation is one of the significant compensatory startup mechanisms. The previous studies show that: up-regulating the lncRNA-NOS3AS expression under hypoxia stimulation can restrain the translation of eNOS and generation of NO, while the mechanism of different degrees of hypoxia that trigger the start and stop of NO-hypoxia signals has not been explained yet. Our previous studies found that: the perception of mild hypoxia (5% oxygen) by brain microvascular endothelial cells is activated by HIF-2α and releases the NO through pulses; it is proved that HuR can directly combine with NOS3AS, and 28 kinds of miRNA that can be reduced under the hypoxia stimulation and can be possibly combined with NOS3AS. Based on those facts, we intend to adopt the model of brain microvascular endothelial cells under mild hypoxia to explore the mechanisms of perceptions of cells on mild hypoxia and pulse release NO; technologies like RNA pull-down and Luciferase reporter assay, etc. are applied to explore the mechanisms for the HuR and miRNA to participate in the regulation of stability of NOS3AS, aiming at make clear the regulating patterns of transcription and post-transcription of NOS3AS/eNOS under mild hypoxia, hoping to reveal the mechanism of cells when they release the NO-hypoxia signals through pulses and providing new drug protection targets and new strategies for the release of cerebral lesions after rapid ascent to high altitude.

高原缺氧失代偿可导致大脑功能障碍，内皮源性NO介导的低氧信号传导是其重要代偿启动机制之一。以往的研究表明：低氧刺激上调lncRNA-NOS3AS表达，进而抑制eNOS翻译及NO生成，但不同程度低氧触发NO-低氧信号启停的机制尚未阐明。课题组前期研究发现：脑微血管内皮细胞感知轻度低氧（5%氧气）是通过HIF-2α活化并脉冲释放NO；证实HuR与NOS3AS具有直接结合作用，并验证得到28个低氧刺激下调且可能与NOS3AS结合的miRNA。籍此，本项目拟采用轻度低氧脑微血管内皮细胞模型，研究细胞感知轻度低氧及脉冲释放NO的机制；应用RNA pull-down、荧光素酶报告基因等技术，研究HuR、miRNA参与调控NOS3AS稳定性的机制；以期明确轻度低氧刺激对NOS3AS/eNOS转录及转录后的调控模式，有望揭示细胞脉冲释放NO-低氧信号的机制，为缓解急进高原脑损害提供药物防护新靶点与新策略。

轻度缺氧初期低氧信号的有效传递可适时启动神经细胞低氧保护反应从而缓解脑缺氧症状，但血管内皮细胞感知不同程度低氧及其产生单个脉冲NO释放的机制尚未完全阐明。本课题研究发现：⑴脑微血管内皮细胞（BMECs）对不同程度低氧的反应有差异；⑵一氧化氮（NO）可体外诱导星形胶质细胞HIF-1α活化；⑶低于5%轻度低氧可刺激诱导BMECs产生单个脉冲eNOS/NO释放；⑷RNA pull-down和质谱鉴定获得9个可能与lncRNA-NOS3AS结合的RBPs，经RIP验证ELAVL1可与其直接结合；⑸miRNAs芯片筛选和qRT-PCR验证获得28个低氧刺激下调且可能与NOS3AS结合的miRNAs；⑹神经元电生理实验证实银杏内酯B可调控缺氧神经元胞内钙稳态平衡。本课题研究明确了低氧刺激BMECs上调ELAVL1的表达和磷酸化，直接结合lncRNA-NOS3AS促进其稳定，并转运至胞浆后结合eNOS mRNA促使其降解，进而产生NO-低氧信号的单个脉冲释放。研究结果揭示了内皮细胞感知轻度低氧及单个脉冲释放NO-低氧信号的机制，丰富了中枢神经系统低氧信号传导路径及其机制的理论基础，为缓解急进高原脑损害提供药物防护新靶点与新策略。