LncRSPH9-4调控致脑膜炎大肠杆菌破坏血脑屏障的作用机制研究

Extraintestinal pathogenic Escherichia coli (ExPEC) possesses the ability to colonize multiple extraintestinal tissues and cause severe infections in human and animals, and has been recognized as an important zoonotic E. coli population which threatening livestock breeding and public health. Meningitic E. coli is an important population of ExPEC strains, but the mechanisms of meningitic E. coli invading host brain and causing blood-brain barrier disruption are poorly understood. Our previous lncRNA transcriptomics analysis revealed an unknown and significantly increased lncRNA, lncRSPH9-4, in response to meningitic E. coli infection. And the lncRSPH9-4 was shown to be positively associated with the expression of vascular endothelial growth factor A (VEGFA), suggesting an involvement of lncRSPH9-4 in meningitic E. coli penetration of the blood-brain barrier through influencing the expression of VEGFA. However, the specific roles of lncRSPH9-4 were unclear. In current study, we are going to characterize the full sequence and features of lncRSPH9-4, and explore the roles as well as the mechanism of lncRSPH9-4 in meningitic E. coli-induced blood-brain barrier disruption. Together, this study will lay foundation for better elucidating the mechanism of meningitic E. coli invasion of host brain, and provide the novel therapeutic targets as well as strategies for protecting blood-brain barrier and preventing the occurrence of bacterial meningitis.

肠外致病性大肠杆菌（ExPEC）能定殖于人和多种动物肠道外组织而引发严重感染，极大危害畜禽养殖和人类公共卫生。致脑膜炎E. coli是ExPEC中十分重要的一类，但其导致宿主血脑屏障破坏的机制仍有待阐明。我们前期转录组学测序发现一条功能未知，并在致脑膜炎E. coli感染后显著上调的LncRNA--LncRSPH9-4。初步分析表明LncRSPH9-4与血管内皮生长因子A（VEGFA）呈现显著的正调控关系，提示该LncRSPH9-4可能通过调控VEGFA参与菌株入侵血脑屏障这一过程，但该LncRNA具体的功能尚不清楚。本研究拟进一步对该LncRNA的全长序列及特征进行解析，通过过表达和干扰/敲除的手段探究LncRSPH9-4在菌株感染破坏血脑屏障中的作用并解析其机制，为全面揭示致脑膜炎E.coli入侵宿主脑组织的机制奠定基础，同时为有效预防细菌性脑膜炎的发生寻找新的药物靶标和防控策略。

细菌性脑膜炎是一种严重威胁生命健康的中枢神经系统感染。致脑膜炎大肠杆菌（E. coli）是最常见的引发脑膜炎的革兰氏阴性致病菌，其致病过程主要包括在外周血持续存在形成菌血症，穿透血脑屏障（BBB）诱导局部炎症，增加BBB通透性并引起严重的中枢炎症反应。近年来有大量研究表明lncRNA参与很多脑部疾病的调控过程，我们前期研究表明致脑膜炎E. coli可诱导人脑微血管内皮细胞（hBMECs）中大量lncRNAs的差异表达，但这些差异lncRNAs在E. coli脑膜炎过程中的作用及调控机制研究鲜有报道。基于前期转录组学测序结果，我们对以lncRSPH9-4为代表的lncRNA在BBB破坏以及诱发中枢炎症中的分子机制展开了系统研究。我们发现，E. coli诱导lncRSPH9-4在hBMECs中上调表达，过表达lncRSPH9-4引起hBMECs细胞通透性增加，导致BBB完整性破坏。通过进一步的RNA-seq分析，我们鉴定到miR-17-5p可以与lncRSPH9-4以及MMP3的3’ UTR区域结合，过量表达的lncRSPH9-4可竞争性吸附miR-17-5p以阻断miR-17-5p对MMP3的下调，由此引起的MMP3上调最终导致hBMECs中紧密连接的破坏及屏障细胞间通透性增大。与此同时，为了更加系统解析lncRNA在E. coli突破BBB中的作用，我们从前期组学结果中选择了另外两条lncRNA—lncC11orf54-1和DDIT4-AS1，探究了其在E. coli感染中对中枢炎症反应的调控作用。在E. coli感染的hBMECs中，lncC11orf54-1会被切割产生成熟体mgU2-30，mgU2-30作为正调控因子参与IRAK1-NF-κB信号通路，促进下游炎性细胞因子的分泌。在星形胶质细胞中，E. coli诱导的DDIT4-AS1可以与其母源基因DDIT4形成RNA二聚体提高DDIT4的稳定性，从而进一步激活下游 NF-κB通路发挥促炎效应。综上，本项目重点阐明了包括lncRSPH9-4在内的lncRNA在致脑膜炎E. coli入侵并破坏BBB和诱导中枢炎症过程中的调控作用及分子机制。上述感染过程中参与调控BBB通透性和中枢炎症反应的关键lncRNA分子将有望作为细菌性脑膜炎诊断及治疗中的重要分子靶标。