LncRNA XIST调控miR-92a促卒中后血管新生和抗炎症损伤的双重作用机制研究

To promote the cerebral angiogenesis and reduce the concomitant severe vascular inflammtory injury after ischemic stroke is a promising target for stroke treatment. Our previous work demonstrated that the α5β1 integrin plays a key role in promoting cerebral angiogenesis in response to cerebral ischemia/reperfusion injury (CI). However, upregulating the integrin α5β1 alone will increase the risk of inflammation after CI. We also found that long noncoding RNA-X inactivate-specific transcript (lncRNA XIST) interacts directly with the microRNA molecule, miR-92a. And the latter negatively regulate cerebral collateral circulation of CI patients and the expression of α5β1 on brain endothelial cells (BECs). It is reported that inhibiting the expression of miR-92a promotes the angiogenesis and reduces the leukocyte influx after myocardial infarction as well as attenuates the endothelial inflammation by upregulating the anti-inflammtion factor- Krüppel-like factor 4 (KLF4). Here, we hypothesize that lncRNA XIST promotes angiogenesis and attenuates vascular inflammtory injury through regulating miR-92a-α5β1/KLF4 after CI. In the current study, we will examine the precise relationship between lncRNA XIST, miR-92a and integrin α5β1/ KLF4, and observe the regulating effect of lncRNA XIST by targeting miR-92a-α5β1/ KLF4 axis to promote the angiogenesis and inhibit the inflammation under cerebral ischemic condition vivo and in vitro. Meanwhile, we also evaluate whether α5β1-ERK/Akt and KLF4-nuclear factor-κB signal pathway is involved in these processes after CI. We first propose that lncRNA XIST interacts with miR-92a-α5β1/KLF4 to regulate the cerebral angiogenesis and vascular inflammation after CI by changing the local microenvironment, which provides a more reasonable therapeutic strategy for ischemic stroke recovery.

血管新生是卒中后修复基础，但一些促血管新生分子常可致血管炎症损伤，因此，亟待探索促血管修复新策略。我们和他人研究发现，miR-92a负向调节促血管新生关键分子整合素ɑ5β1和抗炎因子KLF4，提示抑制miR-92a有促血管新生和减轻炎症反应的双重作用潜能。还发现miR-92a受上游lncRNA XIST调控。由此推测，卒中时上调lncRNA XIST可能通过miR-92a-ɑ5β1/KLF4途径达到促血管新生同时对抗炎症损伤。本项目拟采用缺血性脑卒中模型，运用基因调控、共聚焦成像和免疫沉淀等技术，在细胞和动物水平探究抑制miR-92a或上调lncRNA XIST是否有促血管新生和抑制炎症反应的双重作用；确定lncRNA XIST是否藉调控miR-92a-α5β1/KLF4通路达到促卒中后血管新生和对抗血管炎症损伤；并阐明其内在分子机制。该项目将为卒中后血管保护提供新靶标，为治疗提供新思路。

血管新生是卒中后修复的基础。既往研究主要集中在单纯促进血管新生或抑制炎症反应上，促卒中后血管新生时，较少关注同时并发的炎症损伤。但脑卒中后血管新生常和炎症反应相随。血管新生的同时也存在明显血管炎症损伤。于此，我们提出了促卒中后血管生成新策略：促血管新生同时对抗过度炎症损伤。本项目采用缺血性脑卒中（cerebral ischemic stroke, CIS）模型，结合人体研究，运用基因调控、共聚焦成像和免疫印记等技术，通过调控脑血管内皮细胞或缺血脑区长链非编码RNA X染色体失活特异转录物（long noncoding RNA X inactivate-specific transcript, lncRNA XIST）表达，在细胞和动物水平探究并综合评价其促血管新生和抑制炎症反应的双重作用。结果发现，在CIS患者和体内外缺血模型中，lncRNA XIST的表达在CIS早期呈下降趋势，而在后期表达上调。此外，CIS患者血清lncRNA XIST 水平与神经功能缺损程度呈负相关。进一步的研究显示，lncRNA XIST通过靶向微小核糖核酸-92a（miR-92a）调控促血管生成因子-整合素α5（Itgα5）和抗炎因子-Kruppel样转录因子4（KLF4）的表达。沉默lncRNA XIST可损害CIS后的血管新生，加重脑血管损伤，导致脑缺血小鼠出现更大的梗死灶和更严重的神经功能缺损。机制分析表明，lncRNA XIST分别通过miR-92a/Itgα5或KLF4轴来调控CIS后的血管新生，并减轻脑血管损伤。我们的研究结果表明，lncRNA XIST对CIS具有重要保护作用，血清lncRNA XIST水平可作为预测急性CIS患者预后的潜在生物标志物，本研究为卒中后血管保护提供新的分子靶标，为临床更合理促卒中后血管生成和神经恢复提供新的治疗策略。