LncRNA-MALAT1通过miR-451/IL-6R/STAT3信号通路促进脑出血后血管新生的分子机制

Our previous study has shown that EGb761, an extract of Ginkgo biloba, administration reduces neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage (ICH) mice. While the endogenous mechanisms of angiogenesis in ICH are still unclear. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an endothelial-enriched long noncoding RNA (lncRNA), which has an interesting function to sustain endothelial cell proliferation. MALAT1 upregulation promotes angiogenesis in some physiologic and pathologic conditions. microRNA(miR)‑451 acts as a suppressor for angiogenesis by targeting IL-6R/STAT3 signaling pathway. In this project, we will establish an in vitro model mimicing ICH in primary brain microvascular endothelial cells (BMECs) and an in vivo model of ICH in miR451 knockout mice. Up-regulate or down-regulate MALAT1 and the mRNA transcription or protein expression of miR-451/IL-6R/STAT3 signaling pathway will be measured. The changes about cell cycle, migration and tube formation of BMECs will be detected. We will use modified Nissl staining combined with micro-optical sectioning tomography (MOST, Science 2010) to directly visualize and evaluate the vascular networks in whole brain. In a word, the molecular mechanisms of MALAT1 promoting angiogenesis in experimental ICH via miR-451/IL-6R/STAT3 signaling pathway will be explored from multiple perspectives. MALAT1 may be a potential target of RNA-targeted small molecules drugs for ICH.

本课题组研究发现，给予外源性干预促进血管新生，能减轻脑出血小鼠神经功能损伤，但脑出血后血管新生的内源性机制不清。长链非编码RNA（LncRNA）MALAT1在血管内皮中高表达，其水平升高，可促进血管新生；微小RNA（miR）-451可通过阻断其下游IL-6R/STAT3信号通路，抑制血管新生。本项目拟采用脑微血管内皮细胞和miR-451基因敲除小鼠构建脑出血的体外细胞模型和动物模型，上调或下调MALAT1水平，观察miR-451/IL-6R/STAT3信号通路中各因子水平变化，观察血管内皮细胞的细胞周期、迁徙和成管能力的变化，显微光学切片断层成像系统（MOST，Science 2010）显示全脑微血管，并量化分析。从多水平验证MALAT1/miR-451/IL-6R/STAT3通路是脑出血后调控血管新生的重要机制，MALAT1是调控机制中的关键因子，为靶向RNA的小分子药物研发寻找新靶点。

脑出血（Intracerebral Hemorrhage，ICH）是一种常见的具有高致死率和致残率的神经系统疾病。脑出血后由于血管破裂及血肿占位等给脑组织带来直接损伤，临床治疗时间窗短且治疗手段有限。此外血肿、水肿压迫血管床、破坏血脑屏障、血管活性物质及凝血衍生因子释放可引起血管痉挛及受损、内皮细胞坏死或凋亡等，严重破坏微血管系统，加重脑组织损伤。脑出血后，灶周脑组织血管存在自我修复机制，血管新生是其中重要组成部分。miR-451最早由人脑垂体中提取，参与调控血管内皮功能。本项目以此为研究点，旨在探究miR-451在脑出血后的表达变化、对灶周组织血管新生的影响及其作用机制。我们采用立体定位注射胶原酶法构建小鼠脑出血模型。采用qPCR法观察到脑出血后灶周脑组织中miR-451表达低于假手术组。发现上调miR-451表达可抑制脑出血灶周血管新生及小鼠神经功能恢复；敲除miR-451表达可促进脑出血灶周血管新生及小鼠神经功能恢复。采用hemin处理人脑微血管内皮细胞模拟脑出血微环境。可见hemin处理后hBMECs中miR-451表达降低，而上调、下调miR-451后分别抑制和提高hBMECs的迁移、成管能力。此外，进一步探索miR-451调节血管新生的机制，发现miR-451可与巨噬细胞迁徙抑制因子（MIF）的3’端非翻译区（3'-untranslated region，3'-UTR）结合导致mRNA 的剪切降解，进而使MIF蛋白表达下降。采用双荧光素酶报告基因法验证miR-451与下游蛋白的直接靶点。进一步验证MIF通过调节ERK、AKT蛋白的磷酸化发挥作用。对miR-451敲除小鼠进行脑出血造模后给予腹腔注射MIF抑制剂，发现其可抵消miR-451敲除的促血管新生作用。