非编码环RNA circSirt1与宿主基因SIRT1协同抑制血管平滑肌炎性衰老的机制

We have demonstrated that CKII-SIRT1-SM22 loop evokes a self-limited inflammatory response in vascular smooth muscle cells. Recently, we searched circBase and found that the SIRT1 host gene may produce 11 circRNAs in the human genome. Primary study showed that circ-Sirt1 derived from the SIRT1 gene was markedly reduced in the plasma fraction of peripheral blood in patients with coronary artery disease and in human renal arterial neointima compared with the normal control. We also showed that circ-Sirt1 was significantly decreased in VSMCs treated with TNF-α or PDGF-BB. Overexpression of circ-Sirt1 inhibited the nuclear translocation of NF-κB and c-Myc, decreased the productin of pro-inflammatory factors and cell proliferation. It is unknown that circ-Sirt1 is associated with regulation of inflammaging. We hypothesized that circ-Sirt1 and host gene SIRT1 synergistically inhibits NF-κB activation and vascular inflammaging via direct and indirect mechanisms by inhibiting NF-κB and c-Myc signaling. The molecular network of SIRT1-NF-κB-c-Myc by circ-Sirt1 controls inflammaging and SASP in VSMCs. In the present project, we are going to identify the possible sites of circ-Sirt1 binding to NF-κB or c-Myc, to research the effect of the interaction on the activity of NF-κB or c-Myc and the crosstalking between circ-Sirt1-NF-κB and circ-Sirt1-c-Myc. Furthermore, we are going to predict the potential target miRNAs of circ-Sirt1 and investigate the mechanism of the circ-Sirt1 increase in SIRT1 expression, and elucidate the mechanism by which circ-Sirt1 and host gene SIRT1 synergistically inhibits vascular inflammaging, and determine the role of circ-Sirt1 in the inhibitory regulation of SIRT1-NF-κB-c-Myc on inflammaging. The goal of this project is to demonstrate that the disruption of circ-Sirt1 may be a novel mechanism for NF-κB activation and vascular aging, and is a new biomarker and therapeutic target for atherosclerosis treatment.

我们已证实SIRT1与平滑肌标志物SM22α可构成血管抑炎分子环路。近期发现， 血管平滑肌SIRT1宿主基因高表达非编码环RNA circ-Sirt1，后者在炎症、增殖的平滑肌细胞和人动脉粥样硬化组织及血浆中表达明显下调；过表达circ-Sirt1直接抑制NF-ҝB和c-Myc核转位、上调SIRT1表达、抑制炎性因子生成。我们假设，circ-Sirt1通过直接或间接机制阻抑NF-κB和c-Myc信号转导，与SIRT1共同形成抗炎性衰老和SASP转化的分子网络。本项目拟探讨circ-Sirt1与 NF-κB、c-Myc相互作用的构-效关系和交谈机制，对炎性衰老信号活性的影响；阐明circ-Sirt1上调SIRT1表达的miRNA分子海绵效应和与SIRT1共表达调节机制；揭示circ-Sirt1与SIRT1对炎性衰老的协同抑制和作用模式；为发现血管老化性疾病的新型标志物和干预策略提供研究依据。

我们已证实SIRT1基因外显子2-7产生的环状RNA circ-Sirt1跨空间抑制血管炎症诱导的新生内膜形成。然而，circ-Sirt1在抑制血管炎症和新生内膜形成过程中对VSMC衰老的影响尚未揭示。研究显示，与年轻小鼠比较，老年小鼠颈总动脉组织中circ-Sirt1表达降低，SA-β-gal阳性的衰老细胞在新生内膜中显著增加。敲低circ-Sirt1促进Ang Ⅱ诱导的VSMC衰老；反之，过表达circ-Sirt1抑制Ang Ⅱ诱导的VSMC衰老。使用RNA pull-down结合质谱分析显示，有29个与circ-Sirt1特异性相互作用的蛋白。其中存在抑制细胞周期进程的p53靶分子。使用RNA pull-down、Western blot和RIP进一步证实，circ-Sirt1通过与p53相互作用进而影响其核转位及其衰老相关基因表达，发挥抗衰老作用。基于我们已经证实的circ-Sirt1可上调SIRT1的表达，表型转化的VSMC更易于衰老，进而我们揭示SIRT1抑制表型转化的作用机制。研究显示，SIRT1-Tg VSMCs中cZFP609的表达显著增加；反之，SIRT1-KO 细胞其cZFP609表达明显降低；缺氧或PDGF-BB处理野生型（WT）VSMC则下调SIRT1和cZFP609表达，提示cZFP609可能参与SIRT1相关的VSMC表型转化调节。初步机制研究显示，过表达或激活SIRT1可导致RNA结合蛋白Quaking（QKI）去乙酰化激活，后者介导cZFP609生成；cZFP609可减少PDGFRβ内体再循环利用、促进其内体溶酶体降解，进而衰减PDGF/PDGFR信号传导，维持VSMC处于稳定的收缩表型，与减缓的衰老进程相吻合。总之，circ-Sirt1通过直接作用或通过上调SIRT1表达，形成抑制VSMC表型转化和衰老命运的信号调控网络，维持血管稳态。