内皮祖细胞来源的外泌体lncRNA-AK058003竞争性结合miR-155调控NLRP3在慢性间歇性低氧肾损伤中的作用机制研究

Emerging evidence indicates that chronic kidney disease (CKD) is highly prevalent complication of untreated OSA with symptoms of polyuria and proteinuria. Chronic intermittent hypoxia (CIH) is the foremost pathophysiological change in the process of OSA. We have established a CIH model to investigate the possible mechanism involved in CIH-induced renal injury, but the therapeutic targets still need to be elucidated. Studies have shown that exosomes have superiorities in tissue repair. Previously, we found that endothelial progenitor cells derived exosomes (EPC-Exo) reduced apoptosis and CIH-induced miR-155 levels in injured cells; the results of the chip showed that lncRNA-AK058003 was highly expressed in EPC-Exo upon hypoxia exposure, and the dual luciferase reporter gene further confirmed that the lncRNA-AK058003 binds miR-155. Therefore, we hypothesized that EPC-Exo, which carries lncRNA-AK058003 into injured cells, competitively binds to miR-155 by competitive endogenous RNAs (ceRNAs) pattern, up-regulates the expression of its downstream gene FOXO3a, and inhibits NLRP3 inflammasome, thus promoting tissue repair. Our study applied bioinformatics analysis, RNA-pull down assay, and RNA interference methods to study: the role of ceRNA of EPC-Exo lncRNA-AK058003/miR-155 in CIH-induced renal injury, and its effect on downstream signaling pathways regulated by NLRP3 inflammasome. The results will enrich the target of renal injury from the level of gene regulation, and provide the basis for "cell-free" therapeutic approach in OSA-associated chronic kidney disease.

慢性间歇性低氧（CIH）是OSAHS的重要病理生理机制，是导致肾脏损伤的高危因素。我们前期研究发现CIH通过激活miR-155和NLRP3参与肾损伤的发生；而内皮祖细胞（EPC）来源的外泌体（EPC-Exo）对其具有保护作用。最近我们发现，荧光素酶报告基因证实FOXO3a是miR-155的靶基因；芯片结果表明缺氧微环境中 lncRNA-AK058003在EPC-Exo中特异性高表达，并初步预测了lncRNA存在miR-155的结合位点。由此我们假设，EPC在缺氧刺激下动员入外周血，EPC分泌的Exo携带lncRNA进入受损细胞后，通过竞争性结合miR-155，上调转录因子FOXO3a，抑制NLRP3激活。本项目拟通过构建体内外CIH模型确定EPC-Exo对肾脏的修复作用，结果将从转录调控层面丰富对CIH诱发肾损伤分子机制的认识，为基于靶向修饰非编码RNA的外泌体治疗策略提供科学依据。

慢性间歇性低氧（CIH）是阻塞性睡眠呼吸暂停低通气综合征（OSAHS）的重要病理生理机制。CIH可诱发体内氧化应激失衡、局部和全身产生炎症反应，并且缺氧激活肾内RAS系统、促使交感神经活性增高和肾小球内皮细胞功能障碍，因此CIH是导致肾脏损伤的高危因素。前期研究中，我们发现CIH通过激活miR-155和NLRP3参与肾损伤的发生；内皮祖细胞（EPC）来源的外泌体（EPC-Exo）对其具有保护作用；芯片结果表明缺氧微环境中 lncRNA-AK058003在EPC-Exo中特异性高表达，并初步预测了lncRNA存在miR-155的结合位点。本课题首先通过从人外周血里分离、培养并鉴定EPC，然后提纯且鉴定其外泌体。通过建立慢性间歇性低氧动物模型及间歇性低氧细胞模型，从体内体外水平共同验证外泌体对肾脏的修复作用。随后我们利用病毒载体系统，构建针对lncRNA-AK058003过表达的EPC稳转细胞株，由此获得的EPC-Exo即为被基因修饰过的过表达lncRNA-AK058003的外泌体。利用RT-PCR、ELISA和蛋白免疫印迹方法进一步探究炎症小体及分泌的细胞因子的变化，检测肾组织及HRGEC中miR-155的表达情况。最后利用RNA-结合蛋白免疫共沉淀、RNA pull down和缺失作图法检测分析EPC外泌体转运的lncRNA-AK058003对受体细胞miR-155及靶基因FOXO3a、NLRP3的影响，确立lncRNA-AK058003和miR-155的ceRNA 模式在肾损伤中的调控作用。结果发现EPC在缺氧刺激下动员入外周血，外泌体携带EPC的特异性信息lncRNA-AK058003进入损伤的肾脏细胞后，lncRNA-AK058003通过ceRNA模式竞争性结合miR-155，上调转录因子FOXO3a，抑制炎症小体NLRP3的激活及pro-caspase-1和pro-IL-1β剪切，减少下游多种细胞炎性因子的成熟和分泌，减轻炎症的级联反应，进而促进肾脏自身损伤修复。本课题从LncRNA角度揭示其参与调控炎症损伤的ceRNA新机制，从而探索OSAHS病理生理条件下肾脏损害的特点和分子机制，为相关药物研发提供思路。