LncC11orf54-1在致脑膜炎大肠杆菌介导宿主血脑屏障破坏中的作用机制研究

Meningitis-causing Escherichia coli is capable of inducing blood-brain barrier (BBB) disruption, but the relative mechanisms remain to be elucidated. Our previous study found that the change of a structure and function-unknown LncRNA, designated as LncC11orf54-1, from itself to its mature molecules was induced in meningitis-causing E. coli-infected brain microvascular endothelial cells (BMECs), and LncC11orf54-1 displayed a significantly positive regulation on the phosphorylation of p65 (p-p65), which is the marker of NF-κB activation in BMECs. It is known that the activation of NF-κB in BMECs is directly associated with BBB disruption, suggesting that LncC11orf54-1 may participate in the process of BBB disruption via activating NF-κB during meningitis-causing E. coli infection. However, the role of LncC11orf54-1 and its mechanism in this process are unclear. In this study, we will characterize the structure of LncC11orf54-1 at first. By overexpression different mature molecule-coding areas predicted in LncC11orf54-1, we will identify the mature molecule that has a direct effect on p-p65. After that, the key proteins participating in the process of LncC11orf54-1 regulating p-p65 will be also determined to address the mechanism of LncC11orf54-1 activating NF-κB in BMECs. Finally, we will study the role and mechanism of LncC11orf54-1 in meningitis-causing E. coli-induced BBB disruption through the strategies of LncRNA overexpression, interference and/or knockout. This study will help build a foundation for better understanding the mechanisms of meningitis-causing E. coli-induced BBB disruption.

致脑膜炎大肠杆菌（MCEC）感染能造成宿主血脑屏障（BBB）破坏，但相关机制仍有待阐明。我们前期发现，MCEC感染脑微血管内皮细胞（BMEC）后促进其中一条结构和功能未知的LncRNA——LncC11orf54-1被切割，且该LncRNA对BMEC中NF-κB激活的标志——p65的磷酸化（p-p65）呈现显著的正调控关系。已知BMEC中NF-κB激活与BBB的破坏直接相关，提示LncC11orf54-1可能通过激活NF-κB参与了菌株破坏BBB的过程。本项目拟从解析LncC11orf54-1的序列特征入手，通过分段过表达确定激活p-p65的成熟体，同时寻找参与该过程的关键蛋白并进行验证，从而解析LncC11orf54-1激活NF-κB的机制；最后，通过过表达、干扰/敲除等手段研究LncC11orf54-1在MCEC介导BBB破坏中的作用及机制，为全面揭示MCEC破坏BBB的机制奠定基础。

致脑膜炎大肠杆菌（MCEC）感染能造成宿主血脑屏障（BBB）破坏，但相关机制仍有待阐明。我们前期发现，MCEC感染脑微血管内皮细胞（BMEC）后促进其中一条结构和功能未知的LncRNA——LncC11orf54-1被切割，且该LncRNA对BMEC中NF-κB激活的标志——p65的磷酸化（pp65）呈现显著的正调控关系。已知BMEC中NF-κB激活与BBB的破坏直接相关，提示LncC11orf54-1可能通过激活NF-κB参与了菌株破坏BBB的过程。本项目从解析LncC11orf54-1的序列特征入手，发现LncC11orf54-1主要分布于大脑、肾上腺、卵巢、甲状腺等组织，具有组织特异性且不具有蛋白编码潜能；同时，该LncRNA定位于细胞质并且其过表达可以剂量依赖性地上调p65的磷酸化；此外，生物信息学分析还发现LncC11orf54-1包含两个成熟体mgU2-19和mgU2-30且含有Coilin蛋白的结合位点；在此基础上发现MCEC感染会引起LncC11orf54-1的切割，且切割后产生的成熟体mgU2-30可以诱导p65磷酸化；进一步通过RAP、免疫印迹、质谱分析、RIP、RNA FISH及免疫荧光观察等试验发现LncC11orf54-1及mgU2-30可以结合IRAK1并且可以刺激MCEC感染介导的IRAK1磷酸化，从而激活NF-kB信号，促进细胞因子的表达，加剧中枢炎症反应。本研究阐明了LncC11orf54-1在MCEC介导宿主血脑屏障破坏中的作用机制，对于深入研究细菌性脑膜炎的发生机制具有十分重要的意义。在本项目资助下，发表论文5篇，培养博士研究生1名，项目主持人在项目执行期以博士后身份通过职称评审，晋升副高级职称，并在项目执行期内以正高级职称入职华中农业大学，同时被增选为《中国感染控制杂志》青年编委。