LncRNA RP11-463O9.5对FOXC2的调控及其在胆固醇平衡和动脉粥样硬化发展中的作用

Accumulated evidences show that lncRNAs play an essential role in cardiovascular diseases. However, the effect of lncRNAs on atherosclerosis has not been fully explored. To investigate possible changes in lncRNA expression during atherosclerosis formation, we performed microarray analysis of normal arterial intima and advanced atherosclerotic plaque. The microarray analysis revealed that expression of both lncRNA RP11-463O9.5 and FOXC2 were significantly down-regulated in advanced atherosclerotic plaque when compared with control. Furthermore, bioinformatic analysis showed that the RP11-463O9.5 and FOXC2 were located in the same chromosome. Moreover, FOXC2 expression was enhanced by overexpression of RP11-463O9.5 in macrophages. In addition, cholesterol efflux was enhanced by both RP11-463O9.5 overexpression and FOXC2 overexpression in macrophages. Thus, we hypothesized that atherosclerosis development could be affected by RP11-463O9.5-FOXC2 through regulating cholesterol homeostasis. In this project, we aim to explore the effect and mechanism of RP11-463O9.5-FOXC2 pathway on cholesterol balance and atherosclerosis development by overexpression of RP11-463O9.5 and/or FOXC2. Therefore, our study may provide new scientific evidence to establish RP11-463O9.5-FOXC2 as a new target for regulation of cholesterol homeostasis and prevention of atherosclerosis.

研究表明lncRNA与心血管疾病的发生发展相关，但其对AS影响的研究才刚起步。为了研究lncRNA在AS发生发展中的作用，我们通过基因芯片发现人动脉内膜斑块中lncRNA RP11-463O9.5和FOXC2的表达较人正常动脉内膜中的表达降低。生物信息学分析显示RP11-463O9.5与FOXC2位于同一染色体。预实验结果表明RP11-463O9.5能上调FOXC2的表达，且RP11-463O9.5和FOXC2都能促进细胞内胆固醇流出。因此我们提出RP11-463O9.5可能通过调节FOXC2表达从而调节胆固醇平衡进而影响AS形成。本项目拟通过增强RP11-463O9.5和/或FOXC2的表达，观察RP11-463O9.5-FOXC2对胆固醇平衡和AS形成的影响并探讨相关机制，为确定RP11-463O9.5-FOXC2作为调控胆固醇平衡和防治AS新靶点提供科学依据。

本研究中我们采用FISH和实时定量PCR发现人动脉内膜斑块中lncRNA RP11-463O9.5较人正常动脉内膜中的表达明显增加，进一步体外实验发现，采用氧化低密度脂蛋白处理SMC能浓度依赖性和时间依赖性上调lncRNA RP11-463O9.5表达，过表达lncRNA RP11-463O9.5能明显促进SMC胆固醇蓄积。进一步实验发现SMC过表达lncRNA RP11-463O9.5能明显抑制小凹蛋白（Caveolin-1）的表达，进一步回复实验证明lncRNA RP11-463O9.5通过抑制过氧化物酶体增殖物激活受体γ（peroxisome proliferators-activated receptors γ，PPARγ）的表达调控Caveolin-1的表达。因此，我们提出lncRNA RP11-463O9.5可以介导氧化低密度脂蛋白影响PPARγ和下游caveolin-1的表达进而影响细胞内胆固醇蓄积。另外，我们发现动脉粥样硬化斑块组织中人叉头框蛋白C2（ Human Forkhead box protein C2，Foxc2）较正常动脉组织表降低，采用脂多糖处理HUVEC后，Foxc2表达明显降低而细胞间黏附分子-1（intercellular cell adhesion molecule-1，ICAM-1）的表达明显升高。另外，通过构建Foxc2质粒过表达载体处理HUVEC后，ICAM-1的表达降低，单核细胞粘附内皮细胞数目减少；相反地，通过构建Foxc2小干扰处理HUVEC后，ICAM-1的表达升高，单核细胞粘附内皮细胞数目增加；进一步采用回复实验证明脂多糖能通过影响Foxc2表达进而调控ICAM-1和细胞粘附。因此，本项目我们证明lncRNA RP11-463O9.5和Foxc2能分别通过影响细胞内胆固醇代谢和细胞粘附影响动脉粥样硬化，为lncRNA RP11-463O9.5和Foxc2作为防治动脉粥样硬化的新靶点提供理论依据。