巨细胞病毒源性miR-US33-5p调控血管平滑肌细胞功能改变在主动脉夹层血管壁重构中的作用机制研究

The effects of vascular smooth muscle cell function change regulated by CMV-miR-US33-5p on aortic dissection pathological vascular remodeling and its mechanism of action.Aortic dissection（AD） is an acute macrovascular disease of high mortality and high morbidity. Vascular remodeling refers to alterations in the structure of resistance vessels,which contributing to the pathogenesis of AD. Smooth-muscle cells (SMCs),which is derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel, have a key role in the vascular remodeling. Cytomegalovirus (CMV) infection of SMCs in vivo has been linked to the vascular disease. CMV antigens and nucleic acids have been detected in diseased vessels, and CMV seropositivity is closely associated with the development of arteriosclerosis. It will be important to determine whether CMV or certain substances derived from CMV play a causal role in the pathological vascular remodeling of AD. In our pre-study, we first discover that CMV-miR-US33-5p in the plasma of AD patients had significantly higher expression level than chest pain patients without AD and healthy controls. We hypothesized that CMV-miR-US33-5p would be involved in the vascular remodeling of AD by regulating SMCs function. Firstly, we will detect active CMV infection and pathological changes in AD patients aortic membrane , so as to understand the interaction between CMV infection and AD vascular remodeling . Secondly,we will establish an AD mouse model for studies of CMV infection, and then observe the incidence of AD, pathological changes, specifically the role and mechanism of CMV infection in the vascular remodeling of AD. Thirdly,in vitro studies ,we will detect the change of transcription levels after smooth muscle cells infected with CMV or miR-US33-5p. This study will provide support for the hypothesis that CMV-miR-US33-5p contributes to the pathogenesis of AD and may be an important biological target for the blood-based detection and treatment of AD. It would suggest its potential use for novel strategies of early prevention, treatment and prognostic evaluation of AD.

主动脉夹层（aortic dissection, AD）破裂死亡率极高，血管重构是其发病机制研究中亟待阐明的关键环节，平滑肌细胞功能改变在其中发挥了重要作用。巨细胞病毒可影响平滑肌细胞收缩、分泌等功能。预研究首次发现AD患者血浆中巨细胞病毒miR-US33-5p水平较对照组显著增高，且AD主动脉中膜平滑肌细胞内巨细胞病毒相关抗原呈阳性。那么，巨细胞病毒miR-US33-5p是否通过调控平滑肌细胞功能参与了AD血管重构？其机制如何？本项目拟①通过检测AD患者主动脉中膜巨细胞病毒感染特征与组织病理改变，分析巨细胞病毒与AD血管重构的相关性；②通过构建夹层小鼠模型并对其进行干预，研究巨细胞病毒对AD血管重构的影响；③利用基因转染等技术探索巨细胞病毒miR-US33-5p调控平滑肌细胞功能改变潜在的分子机制。以期为AD发病机制的揭示、寻找新型生物标记物、疾病早期防治等方面提供新思路和策略。

急性主动脉夹层(Acute aortic dissection, AAD)是一种具有极高病死率的灾难性心血管疾病，具有发病突然、进展迅速、死亡率高的特点。目前对其发病机制及治疗仍存在诸多困惑。课题组前期发现miR-US33-5p在动脉夹层患者高表达，抑制人主动脉血管平滑肌细胞 (Vascular smooth muscle cells，VSMC)细胞增殖。通过高通量测序发现miR-US33-5p mimic转染VSMC后EPAS1和SLC3A2表达水平显著下降，且与整合素信号通路密切相关。推测miR-US33-5p通过EPAS1/SLC3A2介导VSMC细胞凋亡，但具体作用机制尚不清楚。为此，课题组通过Q-PCR验证临床样本中miR-US33-5p的表达，发现miR-US33-5p在急性主动脉夹层患者中高表达。然后，利用AD169病毒，miR-US33-5p mimics以及siEPAS1、siSLC3A2分别转染处理VSMC细胞，检测细胞增殖，凋亡和整合素信号通路分子；荧光素酶报告实验和RIP实验检测miR-US33-5p与EPAS1的直接调控关系，以及对SLC3A2的转录调控。结果发现，AD169株病毒感染miR-US33-5p表达上调，VSMC的增殖能力下降，凋亡水平增加，抑制了EPAS1、SLC3A2和GTP-Rac/ Akt/FKA通路的表达；干扰EPAS1、SLC3A2后VSMC的增殖能力下降，凋亡水平增加；miR-US33-5p直接调控EPAS1的表达，进而调控SLC3A2的转录。因此，AD169感染HA-VSMC细胞中miR-US33-5p表达升高，抑制EPAS1的表达，进而抑制SLC3A2的转录，导致GTP-Rac/ Akt/FKA通路活性下调，引发细胞凋亡。通过本项目以期找到治疗AAD的新靶点，为临床治疗提供新依据。