miR-33a在NF-κB促巨噬细胞脂质蓄积中的作用及机制

miR-33a plays important roles in lipid accumulation in macrophages, but the mechanisms that regulate the expression of miR-33a are still not clear. Because miR-33a is coexpressed with its host gene, SREBP-2, we examined the promoter of SREBP-2 using bioinformatics analysis, and found that SREBP-2 promoter includs NF-κB DNA-binding elements. Our preliminary experiments also exhibited that NF-κB activation could up-regulate the expression of miR-33a. Thus we bring up a creative scientific idea that miR-33a is regulated by NF-κB and thereby promoting lipid accumulation in macrophages. Based on the confirmation of NF-κB activation promoting the expression of miR-33a, the project will investigate more deeply the molecular mechanisms of NF-κB regulating miR-33a by luciferase reporter gene assay, EMSA and ChIP etc. Then we will explore the effects of miR-33a on NF-κB promoting lipid accumulation through over expression or endogenous silence it in macrophages. Finally, we evaluate the regulatory role of NF-κB in miR-33a expression at systematic level through establishment of atherosclerosis animal model. In summary, our study will clarify the important role of miR-33a in NF-κB promoting macrophage lipid accumulation, and elucidate the regulatory mechanisms of miR-33a in vivo. These findings will provide new thoughts for deep researching the pathophysiological mechanisms of atherosclerosis and new experimental basis or theoretical foundation for amelioration even treatment of atherosclerosis by targeting miR-33a.

miR-33a在巨噬细胞脂质蓄积中起重要作用，但细胞内调控其表达的机制尚未阐明。我们使用生物信息学分析发现miR-33a的宿主基因SREBP-2启动子区恰含有NF-κB的DNA结合元件，预实验显示NF-κB活化可促进miR-33a表达，因此提出miR-33a受NF-κB转录调控从而促进巨噬细胞脂质蓄积的学术思想。为此本项目拟在确认NF-κB促进miR-33a表达的基础上，采用报告基因分析、EMSA和ChIP等方法深入研究NF-κB调控miR-33a表达的分子机制；通过miR-33a的过表达与内源性沉默，明确其在NF-κB促进脂质蓄积中的作用；建立动脉粥样硬化(As)动物模型，整体上评价NF-κB对miR-33a的调控作用。本项目将阐明miR-33a在NF-κB促巨噬细胞脂质蓄积中的作用及机制，为As的病理生理机制提供新的研究方向，为以miR-33a为靶点防治As提供新的实验依据和理论基础。

泡沫细胞的形成是动脉粥样硬化（As）发病的核心环节, 细胞内脂质过量蓄积是导致泡沫细胞形成的主要原因。微小RNA-33a（miR-33a）位于固醇调节元件结合蛋白-2（SREBP-2）基因中，其主要靶基因是三磷酸腺苷结合盒A1（ABCA1）。miR-33a抑制ABCA1表达，进而影响巨噬细胞脂质流出，促进泡沫细胞形成，并显著增加斑块面积。然而miR-33a在细胞内如何受到调控，还不甚清楚。核因子-kappa B（NF-κB）可抑制ABCA1的表达，导致细胞脂质蓄积；ABCA1基因上并未发现此类直接结合的反应元件。因此我们假设，巨噬细胞中NF-κB活化后很可能是通过与 SREBP-2 启动子区的相应反应元件结合，启动miR-33a的表达，miR-33a 进而抑制ABCA1的表达及脂质流出，导致巨噬细胞脂质蓄积。因此，我们首先明确NF-kB活化与miR-33a表达之间的关系，接着从miR-33a宿主基因SREBP-2启动子调控的角度阐明miR-33a表达受NF-kB调控的分子机制，然后明确miR-33a在NF-kB促巨噬细胞脂质蓄积中的作用，最后在整体水平上评价体内 NF-kB 活化对 miR-33a 表达的促进作用。我们发现，NF-κB活化可促进巨噬细胞脂质蓄积，抑制NF-kB核转移后，巨噬细胞miR-33a与其宿主基因SREBP-2表达明显下降，且巨噬细胞脂质蓄积减轻；促进膜转运体ABCA1表达，可改善脂多糖（LPS）所致的巨噬细胞脂质蓄积，其机制可能与抑制膜受体Toll 样受体4（TLR4）和转录因子NF-κB的表达有关；NF-κB可调控miR-33a及其宿主基因SREBP-2的表达，进而改变ABCA1的表达和巨噬细胞的蓄脂状态；小鼠SREBP-2启动子区域存在潜在的NF-κB作用部位；NF-κB抑制ABCA1/G1表达与SREBP-2和miR-33a有关；体内抑制NF-κB活化，可有效降低miR-33表达，促进ABCA1水平提高，进而降低小鼠的斑块水平。我们的研究在一定程度上揭示了miR-33a在NF-κB促巨噬细胞脂质蓄积中的作用及机制，为As病理生理机制的深入探讨提供新的研究方向，采用药物降低体内NF-κB水平，可能有利于减少人类As的风险，这也为以miR-33a为靶点改善和治疗As提供新的实验依据和理论基础。