循环内皮祖细胞来源的外秘体中lncRNA-PVT5与miR-126互为ceRNA在急性肺损伤中的修复作用及机制研究

Studies have shown that exosomes have superiorities in tissue repair, however, the role and mechanism of circulating endothelial progenitor cells derived exosomes (CEPC-Exo) in acute lung injury (ALI) has not been demonstrated. Previously, we found that CEPC-Exos ameliorated LPS-induced ALI and restored the integrity of pulmonary and upregulated miR-126 level of injured cells; the results of the chip showed that lncRNA-PVT5 was highly expressed in CEPC-Exo, and further confirmed that the lncRNA-PVT5 could bind to miR-126. Therefore, we hypothesized that modification of CEPCs through lncRNA-PVT5 knockdown into injured cells can inhibit the competitive endogenous RNAs (ceRNAs) pattern binding to miR-126 and the expression of its downstream gene PIK3R2, thus promoting lung tissue repair. Our study applied bioinformatics analysis, the dual luciferase reporter gene, RNA-pull down assay, and RNA interference methods to study: the role of CEPC-Exo ceRNAs pattern of lncRNA-PVT5/miR-126 in ALI, and its effect on downstream signaling pathways regulated by PIK3R2. The results will enrich the target of lung injury from the level of gene regulation, and provide the scientific basis for ALI/ARDS stem cell treatment.

外泌体(Exo)在组织修复方面具有优势，我们的前期研究显示循环内皮祖细胞来源的外泌体(CEPC-Exo）能减轻急性肺损伤(ALI)，但作用机制不清；预实验结果表明CEPC-Exo上调靶细胞miR-126水平；芯片结果提示lncRNA-PVT 5在LPS刺激后的CEPC-Exo中特异性高表达，生物信息学软件预测发现lncRNA-PVT 5含有miR-126的结合序列，因此本课题假设，发生ALI时lncRNA-PVT5缺失的CEPC-Exo进入受损细胞，解除了对miR-126的竞争性结合，抑制靶基因PIK3R2的表达，促进肺组织修复；通过荧光素酶报告基因、RNA-pull down等手段研究CEPC-Exo lncRNA-PVT5和miR-126的竞争性内源RNA (ceRNAs)作用模式在ALI中的具体作用，从基因调控层面丰富肺损伤发生的分子靶点，为干细胞治疗提供依据。

研究背景：肺血管损伤是急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）最重要的始动因素。外泌体(Exo)在组织修复方面具有优势，循环内皮祖细胞来源的外泌体(CEPC-Exo）能减轻ALI，但机制不完全清楚。EPCs-exo被证明是细胞间通讯和微环境的重要介质，具有递送短链RNA（miRNA）及非编码RNA（lncRNA）的功能。mRNAs、LncRNA以及环状RNA（circRNA）均含有某些miRNA结合位点，可通过miRNA应答元件（MREs）竞争性结合相同的miRNA，解除或减轻miRNA对靶基因的抑制作用，提高靶基因的表达水平，此竞争性内源RNA (ceRNAs)作用模式参与ALI/ARDS的发病。本项目旨在阐明竞争性内源RNA (ceRNAs)作用模式在ALI/ARDS发病中的作用及机制。结果如下：①EPCs-exo富含miR-126，miR-126被迁移到内皮细胞，通过下调SPRED1基因激活RAF/ERK信号转导通路，以改善内皮细胞的功能。离体研究发现敲除miR-126对内皮祖细胞进行修饰可减弱外泌体的功能。②SCAP患者血清miR-126,miR-27a,miR-146a,及miR-155联合预测SCAP进展为ARDS的价值较高（AUC=0.909）。仅miR-126可潜在预测ARDS的28天死亡率。③circUBXN7的表达在LPS（10μg/mL）诱导的A549、Beas-2B细胞损伤中明显升高；CircUBXN7作为miR-622的海绵，miR-622可逆转circUBXN7对细胞增殖和凋亡的影响。circUBXN7/miR-622可靶向作用于IL6ST，进而通过激活JAK1/STAT3信号通路调控LPS诱导的细胞损伤。④ARDS患者血清中circ_2979表达明显增加；circ_2979与PaCO2、PaO2/FiO2、IL-2R和纤维蛋白原相关。circ_2979有可能作为ARDS的诊断和预后生物标志物。⑤分析了LPS诱导的ALI模型中lncRNA和mRNA的表达情况，揭示了若干重要lncRNA可能是预测ALI发生发展潜在的生物标志物。科学意义：此研究揭示了miRNA、circRNA、lncRNA及其相互作用在内皮祖细胞治疗ALI/ARDS中的新作用及机制，为ALI/ARDS的诊断及干细胞治疗提供新的实验及理论依据。