let-7b在ox-LDL所致人血管内皮损伤中的作用及其信号调控网络研究

The endothelium injury induced by oxidized low-density lipoprotein(oxLDL) is considered the first stage in the pathogenesis of atherosclerosis(AS). It has been discovered by previous studies that oxLDL up-regulates LOX-1 expression and promotes NF-κB(p65) nuclear localization in oxLDL-damaged endothelial cells. The NF-κB(p65) then affects expression of many cytokines, such as ICAM-1 and MCP-1, etc. It is also been demonstrated by our previous study that the microRNA, let-7b, inhibited the expression of LOX-1. We thus hypothesize that let-7b might protect endothelial cells from oxLDL-induced injuries. However the precise effects and mechanisms of let-7b on oxLDL-induced endothelial cell injuries are not clear yet. It is well known that the feedback loop usually exists between microRNAs and transcription factors. MiRNA regulates the expression of transcription factors, such as NF-κB, on the other hand, transcription factors also regulate the transcription of primary miRNA. Thus, we suppose a feedback regulation loop exist between let-7b and LOX-1/NF-κB. This research aims to verify the hypothesis by adopting molecular biological techniques, such as gene transfection and gene interference methods. The effects of let-7b on oxLDL-damaged endothelial cells will be explored, and the cascade of the regulatory signaling pathway will be examined to identify the feedback regulation network between oxLDL, LOX-1, NF-κB and microRNAs. This research is, as to the best of the applicants' knowledge, the first one to investigate the possible of NF-κB/let-7b/LOX-1 signaling loop during the damaging of endothelial cells by oxLDL. Results of the research can provide a new idea and medical target for the therapy of AS.

氧化修饰低密度脂蛋白(oxLDL)可通过上调oxLDL受体1（LOX-1）影响下游NF-κB损伤血管内皮，进而启动动脉粥样硬化（AS）的发生。前期实验证实let-7b可直接抑制LOX-1的表达，但let-7b是否可通过LOX-1而保护内皮免受oxLDL损伤及其机制仍未明确。MiRNA与转录因子间常存在信号调控环路，miRNA可调控转录因子表达，而转录因子也可调控初级miRNA的转录，因此推测可能存在NF-κB/let-7b/LOX-1调控环路。本课题拟采用基因转染和干扰等分子生物学技术，研究let-7b对内皮的保护作用，探讨let-7b与AS的关系，深入研究NF-κB/let-7b/LOX-1调控环路，及信号转导途径。本课题首次在oxLDL损伤的内皮细胞中探讨let-7b的作用及NF-κB/let-7b/LOX-1调控环路，研究成果可为AS防治提供新思路和新靶点。

心脑血管疾病是目前世界上发病率和死亡率最高的疾病，动脉粥样硬化（AS）诱发冠心病、脑卒中、心肌梗死等心脑血管疾病的独立危险因素。深入研究AS的发病机制及调控机制，为治疗AS寻找新的靶点及治疗方法，已成为当前研究的重要目标。目前认为血管内皮损伤是AS的启动因素，而高血脂引起的氧化低密度脂蛋白（oxLDL）升高，是内皮损伤的关键因素。对抗oxLDL，保护血管内皮将会在源头上抑制AS的发生发展。oxLDL可通过上调oxLDL受体1（LOX-1）影响下游NF-κB损伤血管内皮，进而启动AS的发生。let-7家族成员a/b/c可直接抑制LOX-1的表达，但let-7a/b/c是否可通过LOX-1而保护内皮免受oxLDL损伤及其机制仍未明确。. 本研究研究内容有：一，观察了let-7a/b/c对oxLDL损伤的血管内皮细胞结构和功能的影响。观察在let-7a/b/c过表达的情况下，正常对照组和oxLDL损伤组细胞形态和功能的变化；二，观察了核转录因子NF-κB与let-7c的相互调控关系。本研究构建了NF-κB表达载体，并利用NF-κB抑制剂PDTC，采用实时定量PCR技术，观察NF-κB过表达和抑制时，细胞内let-7c的表达变化；采用Western-blot技术，观察let-7c过表达和抑制时，细胞内NF-κB的表达变化，揭示NF-κB与let-7c的相互调控作用。并构建let-7c启动子上游-0.5kb、-1kb、-1.5kb长度的双荧光报告基因，检测NF-κB对荧光报告基因表达的影响；采用免疫共沉淀法（ChIP），与凝胶迁移实验（EMSA），进一步验证NF-κB与所预测结合位点的相互关系。三，研究了let-7c/LOX-1/ROS/p38MAPK/NF-κB信号转导途径。.本研究研究结果发现，在oxLDL损伤的内皮细胞中，let-7a、let-7b、let-7c的表达被抑制，并呈时间和浓度依赖性；2）let-7a、let-7b、let-7c过表达可以对抗oxLDL所致的内皮细胞凋亡和分泌功能改变；3）let-7a、let-7b、let-7c保护内皮的作用与LOX-1/ROS/p38MAPK/NF-κB通路及LOX-1/ROS/PKB /eNOS通路有关。双荧光报告基因、EMSA和ChIP实验证明NF-κB p65和p50可直接作用于miR-99a/let-7c的启