一种新的长非编码RNA抑制血管内皮细胞凋亡的分子机制研究

It has been well accepted that vascular endothelium is critically involved in the maintenance of vascular homeostasis in health. Therefore, it is very important to discover the new apoptosis inhibitor or pathway in vascular endothelial cells for vascular homeostasis and remodeling. In our previous study, we found a new long noncoding RNA—AF007131 that inhibited vascular endothelial cell (VEC) apoptosis, but its action mechanism is unknown. By using bioinformatics programs, we known that the new long noncoding RNA may interact with miRNA-4707-5p and miRNA-4767 more strongly, and these two miRNAs may target anti-apoptosis factors API5(Apoptosis inhibitor-5）and BCL2L12 (Bcl2-Like 12，Bcl2L12) respectively. In our experiments, we demonstrated that overexpression of AF007131 down regulated miRNA-4707-5p and miRNA-4767, and positively regulated API5, BCL2L12 and its neighbor gene-BCL2L10; whereas, knockdown of AF007131 up regulated miRNA-4707-5p and miRNA-4767, and down regulated API5, BCL2L12 and its neighbor gene-BCL2L10 by using Quantitative real-time PCR. Based on these results and the latest literatures, we raise a hypothesis that, on the one hand, AF007131, which may interact with miRNA-4707-5p and miRNA-4767, up regulate API5 and BCL2L12 and then inhibit apoptosis; on the other hand, AF007131 may positively regulate BCL2L10 by epigenetic modification, further inhibit apoptosis. We will demonstrate our hypothesis by using binding site mutation, RNA interference, gene overexpression and luciferase reporter system, RNA pull-down，ChIP, RNA ChIP and other molecular/cell biological methods. By this study, we will provide new evidence for explaining the mechanism of this long noncoding RNA in control of vascular endothelial cell apoptosis, and discover new targets for prevention of vascular diseases.

血管内皮细胞凋亡是导致血管稳态失衡和重构的重要因素，因此，发现抑制血管内皮细胞凋亡的新因子及通路对控制血管稳态和重构至关重要。在预实验中，我们发现了一种抑制血管内皮细胞凋亡新长非编码RNA—AF007131，但是,其作用机制尚未搞清。生物信息学分析表明，它与miRNA-4707-5p和miRNA-4767有很强的结合能力，其靶基因分别是凋亡抑制因子API5和BCL2L12；过表达或敲低AF007131分别明显下调或上调这两个miRNAs，并上调或下调两者的靶基因，同时还能正调控其临近基因BCL2L10。因此，提出科学假设：AF007131一方面通过与miRNA结合进而上调其靶基因API5和BCL2L12；另一方面，通过改变BCL2L10其上的表观遗传修饰进而正调控BCL2L10，从而抑制细胞凋亡。我们拟利用培养的内皮细胞和ApoE-/-小鼠模型，证明该假设,为防治动脉硬化提供新靶点和线索。

长非编码RNA（lncRNA）是一类长度超过200nt，无编码能力的转录产物。lncRNA可以在表观遗传修饰水平、转录水平和转录后水平等多个层次调控基因表达，从而在分化、增殖、凋亡、迁移和多能干细胞再生等多个生理过程中起到重要作用。血管内皮细胞（VECs）凋亡与动脉粥样硬化、血栓形成和斑块损伤等多种心血管疾病相关。目前，血管内皮细胞凋亡相关研究主要集中在蛋白水平上，直接与血管内皮细胞凋亡相关的lncRNA的研究较少。.血清和生长因子饥饿是诱导人脐带静脉内皮细胞（HUVECs）凋亡的良好模型。在之前的研究中，我们发现苯并噁嗪衍生物ABO可以提高血清和生长因子饥饿下的HUVECs存活率；在ApoE-/-小鼠中，ABO可以抑制oxLDL诱导的VECs凋亡；但目前ABO抑制凋亡的机制尚不清楚。因此，我们以ABO作为VECs凋亡抑制剂，寻找了抑制VECs凋亡的新因子和新通路。.在本项目中我们发现了一种新的抑制血管内皮细胞凋亡lncRNA(CERNA1)。研究表明， 在细胞质中，CERNA1通过竞争性内源RNA途径抑制血管细胞凋亡，CERNA1靶向两个miRNA（miRNA-4707-5p和miRNA-4767），进而上调两个凋亡抑制因子API5和BCL2L12的表达，从而抑制细胞凋亡。在细胞核中CERNA1通过表观遗传修饰调控途径促进血管内皮细胞凋亡，CERNA1可以对邻近基因BCL2L10的启动子区进行表观遗传修饰（降低DNA甲基化水平，提高H3K4me3和H3K9ac的组蛋白修饰水平），进而增加凋亡促进因子BCL2L10的表达，从而促进细胞凋亡。ABO不仅上调CERNA1的表达，还可以改变CERNA1在核质中的分布比例，提高CERNA1在细胞质中的比例，两个方面共同作用进而抑制细胞凋亡。在ApoE-/-小鼠中过表达CERNA1可以通过提高斑块内平滑肌细胞和巨噬细胞的API5蛋白水平进而减少细胞凋亡，从而提高动脉粥样硬化斑块的稳定性。小分子化合物ABO以TIA-1依赖的方式促进CERNA1的表达。.总之，我们利用ABO发现了控制血管内皮细胞凋亡的新因子和信号通路，这对控制血管稳态和重构具有重要意义，同时也为防治血管疾病的新药物研发奠定基础。