Lp-PLA2基因的表观遗传修饰在动脉粥样硬化病变进展中的作用及机制

Lipoprotein-associated phospholipase A2 (Lp-PLA2) has emerged as a potential translational target in coronary heart disease (CHD)therapy. In previous studies supported by NSFC, we found enzymatic expression of Lp-PLA2 was up-regulated within the atherosclerotic lesion. This was associated with plaque progression and instability. Further, suppression of Lp-PLA2 retarded atherosclerosis progression and decreased plaque inflammation. However, the precise mechanism of transcriptional control of Lp-PLA2 gene expression remains uncertain, which may play an essential and fundamental role in the pathogenesis of atherosclerosis. Relevance in recent years, increasing attention has been placed on the role that altered epigenetic states might play in atherosclerosis development, partly because unlike genetic changes, epigenetic changes are potentially reversible. Epigenetic events act as the regulatory switches of gene transcription and have pivotal clinical significance. Thus, the goal of the project is to characterize the epigenetic pattern changes of Lp-PLA2 gene expression that take place in atherosclerotic lesion and underlying mechanisms. We hypothesize exposure to specific stimuli (e.g. cytokines) or risk factors will induce the certain enzymes involved in epigenetic events (e.g., DNMTs for DNA methylation, HMTases and HDACs for histone modifications.) altering，leads to abnormal epigenetic status of Lp-PLA2 and deregulation of gene expression, finally to atherosclerosis progression. We will extend our previous work by performing a more detailed analysis of DNA methylation and histone modifications and Lp-PLA2 gene expression in atherosclerosis. First, we will observe the epigenetic pattern of Lp-PLA2 in human carotid plaques, as well as the association between the dynamic epigenetic changes and atherosclerosis progression in porcine model of atherosclerosis. Several methods, including MeDIP-qPCR, BSP-sequencing and ChIP, will be used to detected DNA methylation and histone modification state of Lp-PLA2. And then, we will identify the regulatory mechanism of Lp-PLA2 epigenetic modification in human THP-1,murine RAW264.7 cell line and human peripheral blood monocyte derived macrophage by loss-of-function/gain-of-function of the key modulators involved in. Since a causal link connecting epigenetic events to altered Lp-PLA2 gene expression and atherosclerosis initiation/progression is yet to be established, identifying whether epigenetic alterations act as the source of Lp-PLA2 gene expression in atherosclerosis would be a critical advance for the field of atherosclerosis pathogenesis, and highlight potential therapeutic interventions based on epigenetic mechanisms.

脂蛋白相关性磷脂酶A2(Lp-PLA2) 是一个具有重要临床转化价值的动脉粥样硬化(AS)防治靶点。在前两项国家自然科学基金资助下，我们发现AS病变中Lp-PLA2表达增加与病变严重程度呈正相关。由于对Lp-PLA2表达调控仍未充分了解，限制了以Lp-PLA2为靶点的抗AS药物研发。本项目从表观遗传修饰入手，在人AS斑块、小型猪AS模型和多种因素刺激的单核/巨噬细胞上，应用免疫印迹、定量PCR、亚硫酸氢钠修饰后测序、ChIP、免疫共沉淀、激光共聚焦显微镜、双荧光素酶报告基因等方法，检测Lp-PLA2表达及其主要表观遗传修饰、表观遗传修饰转移酶、转录元件结合等的变化，并通过基因过表达和沉默技术双向调节Lp-PLA2的表观遗传修饰，进一步观察上述指标的变化，从而明确Lp-PLA2表观遗传修饰与AS病变进展的关系，并揭示AS时调控Lp-PLA2表达的表观遗传机制和影响因素。

脂蛋白相关性磷脂酶A2(Lp-PLA2) 是一个具有重要临床转化价值的动脉粥样硬化(AS)防治靶点，AS 病变中Lp-PLA2 表达增加与病变严重程度呈正相关。由于对Lp- PLA2 表达调控仍未充分了解，限制了以Lp-PLA2 为靶点的抗AS 药物研发。本项目以表观遗传调节的DNA甲基化调控基因表达为切入点，利用全基因组甲基化芯片，针对Lp-PLA2基因深入分析。我们首先利用全基因组甲基化芯片对人颈AS斑块和正常血管进行DNA甲基化测定，在斑块中发现了9577个低甲基化位点。利用ClueGo、DAVID等生物信息数据库分析有差异表达的甲基化基因的生物学功能，发现斑块中低甲基化基因几乎都参与炎症反应，提示炎症反应受表观遗传调控，其中有124个低甲基化基因在破裂斑块、稳定斑块、早期斑块及晚期斑块中均表达上调。其中，Lp-PLA2启动子甲基化位点个数显著减少，斑块中Lp-PLA2表达明显增多，提示Lp-PLA2与AS存在重要关联。进一步我们培养THP-1细胞并给予甲基化酶抑制剂，利用实时定量PCR、蛋白免疫印迹和酶联免疫吸附检测方法，分别检测Lp-PLA2的THP-1细胞中mRNA水平和蛋白水平以及THP-1细胞上清液的蛋白水平，证实低甲基化水平能够促进Lp-PLA2的上调，提示表观遗传中的甲基化变化可以调节Lp-PLA2的表达水平，与AS病变中甲基化变化的结果一致。最后，我们利用亚硫酸氢盐修饰后测序的方法，检测甲基转移酶抑制剂处理后的细胞甲基化水平，发现Lp-PLA2启动子区和第一外显子甲基化减少；进一步在去甲基化条件下利用染色质免疫沉淀技术，观察转录因子与相关位点的结合状态，发现Sp3结合的DNA序列是增加的，明确了甲基化调节Lp-PLA2的相关分子机制。. 通过相关实验，本课题组证实了表观遗传调节DNA甲基化在AS中的关键作用，重要基因Lp-PLA2的调节与AS的内在关联。Lp-PLA2的表达受到启动子和第一外显子的甲基化调控，Sp3是调控的关键。在动脉粥样硬化疾病的过程中，通过低甲基化Lp-PLA2启动子，使得Lp-PLA2表达增加，进而促进AS的发生发展。因此，本研究揭示动脉粥样硬化疾病过程中调控Lp-PLA2表达的表观遗传机制和影响因素，为开辟基于Lp-PLA2的新型抗动脉粥样硬化治疗策略提供理论和科学依据。