Abstract：
The initiation and progression of atherosclerotic plaque caused by abnormal lipid metabolism is one of the main causes of atherosclerosis (AS). Lipid droplet accumulation has become a novel research pointcut for AS treatment in recent years. In AS patients, miR-135b level was up-regulated relative to the normal cases, which showed negative correlations with the levels of Semaphorin 3A (SEMA3A) and circZNF609, separately. The U937-derived macrophages were cultured with ox-LDL to establish AS models in vitro. After that, the lipid accumulation, inflammation, mitochondrial dysfunction and cell death were evaluated by ORO, ELISA, RT-qPCR, western blot, JC-1 and FCM assays respectively. Transfection of the circZNF609 expression vector notably declined lipid accumulation, attenuated inflammation, reduced mitochondrial dysfunction and inhibited cell death in ox-LDL-stimulated cells. The direct binding of miR-135b to circZNF609 in vitro was confirmed using RIP assay, and SEMA3A expression was up-regulated by circZNF609 overexpression. After manipulating the endogenous expressions of circZNF609, miR-135b and SEMA3A, the above damages in ox-LDL-stimulated cells were rescued by inhibition of miR-135b expression and overexpression of circZNF609 or SEMA3A. Besides, the AS mice model was built to demonstrate the excessive lipid accumulation, increasing inflammation and cell death in AS pathogenesis according to the results of HE staining, ELISA and IHC assays, while these damages were reversed after overexpression of circZNF609 or SEMA3A. In AS models, overexpressed circZNF609 prevents the AS progression through depleting miR-135b expression and subsequent up-regulation of SEMA3A expression to overwhelm lipid accumulation, mitochondrial dysfunction and cell death.
摘要：
脂质代谢异常惹起的动脉粥样硬化斑块的发生和进展是动脉粥样硬化(AS)的主要缘由之一。近年来，脂滴积累已成为AS治疗的新研究重点。在AS患者中，miR-135b水平相对于正常病例上调，分别与信号素3A（SEMA3A）和circZNF609水平呈负相关。U937来源的巨噬细胞与ox-LDL培养以建立体外AS模型。之后,脂质积累，炎症,ORO评估了线粒体功能障碍和细胞死亡，ELISA,RT-qPCR,westernblot,JC-1和FCM分别测定。circZNF609表达载体的转染显著降低了脂质积累，减轻炎症，减少线粒体功能障碍并抑制ox-LDL刺激细胞的细胞死亡。使用RIP测定证实了miR-135b与circZNF609的体外直接结合，和SEMA3A表达被circZNF609过表达上调。在操纵circZNF609，miR-135b和SEMA3A的内源性表达后，通过抑制miR-135b表达和circZNF609或SEMA3A的过表达来挽救ox-LDL刺激细胞中的上述损伤。此外,建立AS小鼠模型以证明脂质过度积累，根据HE染色结果，AS发病机制中炎症和细胞死亡增加，ELISA和IHC分析，而这些损害在circZNF609或SEMA3A过表达后被逆转。在AS模型中，过度表达的circZNF609通过耗尽miR-135b表达和随后上调SEMA3A表达以压倒脂质积累来阻止AS进展，线粒体功能障碍和细胞死亡。
