长链非编码RNA PICALM-IT1调控小窝蛋白1在肝肺综合征PMVECs内皮间质转化中的机制研究

Hepatopulmonary syndrome (HPS) is highly risk and hardly to prevented. Intrapulmonary vascular dilation is the basis of the pathophysiology. Cav1 is one of the important part of the cytomembrane of the lipid raft, it can promote the endothelial mesenchymal transition (EndMT) of PMVECs. However, the regulation mechanism is not clear. Our previous study have certified that miR144-3p may reduce the expression of Cav1, and have the key role in inhibiting normal EndMT. Further, long noncoding RNA PICALM-IT1 may regulate miR144-3p level in the PMVECs. So, we hypothesis that “The expression of LncRNA PICALM-IT1 is increased in the HPS rat lung, and formatted lots of pseudogenetic transcription to binding with miR144-3p and then upregulate synthesis of Cav1 and promote IPVD of HPS”. For this purpose, Luc reporter system, site-specific mutagenesis, Isothermal Titration Calorimetry and Mapping RNA interactome in vivo methods to clarify how to regulating of PMVECs EndMT by PICALM-IT1. The result may explain how PICALM-IT1 promote PMVECs EndMT, with highly theoretical significance and potential application value.

肝肺综合征(HPS)致死率高、防治困难。肺微血管扩张是其病理学基础。小窝蛋白1(Cav1)是细胞膜脂筏的重要组分，可促进肺微血管内皮细胞(PMVECs)间质转化，但其调控机制不详。前期研究发现，miR144-3p可能负调控Cav1，对抑制正常PMVECs间质转化至关重要，而长链非编码RNA PICALM-IT1可能调控miR144-3p的水平。据此提出“HPS病理状态下，PICALM-IT1通过与miR144-3p结合，降低了miR144-3p对Cav1的抑制，从而促进PMVECs内皮间质转化，加速了HPS病理性肺微血管扩张”。为此，本项目拟采用双荧光素酶报告系统、定点突变、等温热滴定、活体RNA互作组谱等手段分析PICALM-IT1、miR144-3p与Cav1相互竞争性结合。本研究有望初步阐明PICALM-IT1促进PMVECs间质转化的分子机制，具有重要的理论意义和潜在的应用价值。

肝肺综合征(HPS)致死率高、防治困难。肺微血管扩张是其病理生理学基础。本课题利用转录组测序技术发现了一个在HPS病理状态肝脏中高量表达的新的长链非编码RNA，将其命名为PICALM-AU1。结合荧光原位杂交技术对其表达模式分析发现，它主要表达在肝脏胆管上皮细胞中，且在HPS病理状态下表达量上调。亚细胞定位发现它即存在于细胞核 中也存在于细胞质中，结合FISH结果，我们发现PICALM－AU1主要合成的地方有大量的外泌体聚集，这暗示该分子可能通过外泌体的形式分泌到肺脏，进而发挥作用。而大鼠数据和HPS病人的数据确证了这一假设；对其功能分析发现，PICALM－AU1可通过调控EndMT而促进肺微血管的增生。进一步，利用生物信息学结合细胞转染、双荧光素酶等实验证实，PICALM-AU1通过抑制miR144-3p对ZEB1的负调控，从而促进了PMVECs间质转化，加速了HPS病理性肺微血管扩张。本研究初步阐明了PICALM-AU1促进PMVECs间质转化的分子机制，具有重要的理论意义和潜在的应用价值。