HIV-1 Tat增强P2Y2R信号对神经元损伤的调节：lncRNA的作用

Human immunodeficiency virus type 1 (HIV-1) Tat, as a regulating protein, is essential for the pathogenesis of HIV-associated neurocognitive disorder (HAND) that is characterized by abnormal astrocytosis resulted from inflammatory factors. Accumulating reports show that purinergic P2Y2 receptor (P2Y2R) signaling plays a unique role in the neuron damage and function regulated by the astrocytes. Recently, our results indicate Tat can up-regulate P2Y2R expression both in primary astrocytes (in vitro) and the brain from Tat-induced HAND mice (in vivo). However, the mechanism and role of P2Y2R expression induced by Tat are less-known. The non-coding RNAs (lncRNAs) provide a new view of the gene expression regulation. Our studies represent that the lncRNA Gm14047 displays sequence similarity to antisense of IL-1 beta. Of importance, both the expression of Gm14047 and P2Y2R is up-regulated at the same time and display positive correlation in the astrocytes stimulated by Tat protein, which suggests that the lncRNA Gm14047 is a positive regulator of P2Y2R that has significantly effect on the neuron damage. In this project, we plan to elucidate the role of the lncRNA Gm14047 in regulation of the neuron damage and the regulation mechanism of P2Y2R expression from the epigenetics in HAND mice. If successful, this project will broaden our vision on the acknowledgements of P2Y2R signaling and the mechanism of the neuron damage in the HAND.

HIV-1 Tat介导的炎性因子持续存在和由其引发的星形胶质细胞异常活化参与HAND发生发展。嘌呤受体P2Y2R参与胶质细胞对神经元存活及功能调控。我们发现Tat可促进星形胶质细胞P2Y2R表达，但Tat上调P2Y2R表达的机制及其作用尚待探索。长链非编码RNA（lncRNA）是基因表达调控的新着眼点，我们发现lncRNA Gm14047与IL-1β反义链同源；Tat刺激的星形胶质细胞中Gm14047与P2Y2R表达同步上升，二者呈正相关。提示该lncRNA是P2Y2R的正调控因子，可调控P2Y2R表达影响神经元损伤。本项目拟应用慢病毒Tat制备的HAND模型，明确Gm14047在调控神经元损伤中的作用，从表观遗传学探索其调控P2Y2R表达机制，拓宽对P2Y2R的认识，深入解析HAND中神经元损伤的机理，为发展基于HAND发生发展的神经保护提供新策略和研发理想的治疗药物提供新靶点。

目的：HIV脑病（HIV-associated neurocognitive disorder，HAND），是一类HIV感染后诱发认知和运动功能紊乱为特征的神经退行性疾病，以星形胶质细胞异常活化、神经元缺失为HAND的主要病理特征。加强HAND发病机制研究，发现能够延长神经元存活、保存神经元功能的关键靶标，研发理想的神经保护剂已经成为当代神经科学最重要的研究目标之一。.方法：.1. Western blot技术检测Tat蛋白处理小鼠原代星形胶质细胞P2Y4R蛋白及下游信号通路PI3K/AKT、MAPK（ERK、P38和JNK）等信号通路表达变化；.2. 基因芯片检测Tat重组蛋白处理小鼠原代星形胶质细胞mRNAs表达谱变化；.3. 免疫荧光和Western blot检测p65在细胞中的定位情况；.4. 应用 CBA技术检测NF-κB抑制剂Bay11-7082（Bay）和500 nM BRD4抑制剂JQ1预处理后上清中趋化因子IP-10、MCP-1、MIP-3α以及EOTAXIN（CCL11）的分泌情况，qRT-PCR检测趋化因子及炎症因子的转录情况；.5. TUNEL染色观察IQ1对神经元凋亡的影响，集总RNA，行RNA 测序并进行数据分析，lncRNA基因芯片检测lncRNA表达谱变化；.6. qRT-PCR检测lncRNAs和CXCL-趋化因子基因的表达，RIP技术研究AK017645对CXCL10表达调控的机制。.结果：HIV-1 Tat蛋白促进原代星形胶质细胞细胞因子的释放是通过P2Y4R信号途径介导的；HIV-1 Tat刺激原代星形胶质细胞、神经元及小胶质细胞后能够导致细胞的活化促使炎性细胞因子的大量释放，其中包括IL-6、TNF-α、MCP-1 、IP-10 、MIP-3α等，使用BRD4和NF-κB抑制剂后发现相关炎症细胞因子的释放明显被抑制，说明溴结构域蛋白BRD4在HIV-1 Tat介导的神经炎症中起重要作用。.结论：HIV-1 Tat诱导小鼠原代星形胶质细胞lncRNAs差异表达，其中第5号染色体CXCL-趋化因子（CXCL3/CXCL1/CXCL2和CXCL9/CXCL10/CXCL11）基因簇5’端Uba6基因的AK017645（sense overlap lncRNA）结合乙酰化的NF-κB，从而促进CXCL10的表达。