LncRNA AC096664.3-UGP2 途径介导ox-LDL对泡沫细胞和AS 的影响及机制研究

It is well established oxidized low-density lipoprotein (ox-LDL) promotes foam cell formation and plays a crucial role in the initiation and progression of atherosclerosis, and long non-coding RNA (lncRNA) is closely related to the foam cell formation and atherosclerosis development. Our preliminary data showed both lncRNA AC096664.3 and UGP2 expression levels were significantly up-regulated in ox-LDL-induced macrophage-derived foam cells as compared to non-ox-LDL treated macrophages by microarray analysis, real-time quantitative PCR and western blot. Moreover, we demonstrated UGP2 expression levels were markedly up-regulated by overexpression of AC096664.3 in macrophages and accumulation of cholesterol was significantly enhanced by treatment with both overexpression of AC096664.3 and overexpression of UGP2 in macrophages. Thus, we hypothesize that foam cell formation and atherosclerosis development can be affected by ox-LDL through inducing AC096664.3 and UGP2 expression. In this study, we aim to explore the effect and mechanism of ox-LDL-AC096664.3-UGP2 pathway on the foam cell formation and atherosclerosis development by enhancing or inhibiting expression of AC096664.3 and/or UGP2. Therefore, our study may provide a new scientific evidence to establish ox-LDL-AC096664.3-UGP2 pathway as a new target for regulation of cholesterol balance and prevention of atherosclerosis.

本课题组和其他学者的研究表明ox-LDL可促进泡沫细胞形成和AS进展，lncRNA与泡沫细胞形成和AS进展密切相关。我们采用基因芯片分析巨噬细胞和巨噬细胞源性泡沫细胞中差异表达的基因，发现巨噬细胞经ox-LDL处理后lncRNA AC096664.3和UGP2表达上调。进一步验证发现ox-LDL能上调AC096664.3和UGP2的表达，过表达AC096664.3能上调UGP2的表达，过表达AC096664.3和UGP2都能促进细胞内胆固醇蓄积。因此我们提出ox-LDL可能通过增加AC096664.3表达从而上调UGP2表达进而促进泡沫细胞形成和AS进展。本项目拟通过增强/抑制AC096664.3和/或UGP2表达，观察ox-LDL-AC096664.3-UGP2途径对泡沫细胞形成和AS进展的影响并探讨相关机制，为确定该途径可作为调控胆固醇平衡和防治AS新靶点提供科学依据。

项目背景：ox-LDL刺激巨噬细胞后LncRNA AC096664.3表达下降，表明LncRNA AC096664.3对调控细胞内脂质稳态具有重要作用，但其在平滑肌细胞中的作用尚不清楚。细胞糖代谢除了起到维持细胞基础能量消耗的作用外，还可能通过影响内皮细胞功能参与动脉粥样硬化等心血管疾病的发生发展过程，但具体机制不清楚。动脉内脂质的高分辨率成像对获得其在动脉粥样硬化中作用的基础知识具有重要价值，但尚缺乏理想的探针。.研究内容：50μg/mL的ox-LDL 刺激VSMCs后发现LncRNA AC096664.3表达下降至对照组的80%，并且调控胆固醇流出相关蛋白ABCG1和调控ABCG1相关蛋白PPAR-γ的基因和蛋白质水平表达也显著下降，说明ox-LDL抑制LncRNA AC096664.3表达从而抑制PPAR-γ表达进而抑制ABCG1的表达，使细胞内胆固醇堆积。.UGP2在人动脉粥样硬化斑块组织的表达较正常内膜组织显著下调。在细胞水平验证 UGP2能抑制内皮细胞凋亡、抑制cleaved caspase-3的表达和降低细胞内ROS的水平：对照细胞的凋亡率为4.2±0.7％，而UGP2低表达细胞的凋亡率为13.3±2.0％，UGP2低表达细胞的ROS水平增加至144±12.4％；UGP2上调的细胞中，ROS水平下降至对照细胞的66±7.1％，H2O2处理实验证明UGP2通过氧化应激调控细胞凋亡。通过染色质免疫共沉淀的方法发现TP53正向转录调控UGP2。最后通过构建UGP2 +/- LDLR -/-和UGP2 +/+ LDLR -/-小鼠验证UGP2能够抑制细胞凋亡、抑制动脉粥样硬化斑块进展。.结论：ox-LDL通过LncRNA AC096664.3/PPAR-γ/ABCG1途径降低ABCG1表达使细胞内胆固醇堆积。关键的糖原代谢酶UGP2是TP53的新型直接靶标，UGP2可以通过减少ROS产生和降低cleaved caspase-3的水平来调控细胞凋亡。