Role of miR-146a in Abdominal Aortic Aneurysm

摘要 腹主动脉瘤是腹主动脉的永久性扩张，死亡率高 破裂后大于80%。主动脉血管平滑肌细胞（SMC）在维持主动脉 结构完整性和功能，富含SMC的主动脉内侧稳定性在AAA中被高度破坏。目前， 除了外科手术外，没有其他治疗方法可以阻止AAA的进展和破裂。 因此，迫切需要确定新的策略，以开发有效的，非手术的， 治疗学MicroRNA-146 a（miR-146 a）是一种众所周知的炎症和自身免疫调节因子， 在AAA患者的动脉瘤组织中表达。然而，miR-146 a在SMC稳态和细胞增殖中的作用可能与其在细胞增殖中的作用有关。 AAA仍有待探索。在初步研究中，通过原位杂交，我们观察到miR-146 a是 在人和小鼠AAA的富含SMC的主动脉培养基中上调; miR-146 a缺陷显著促进 在与赖氨酰氧化酶抑制剂共同给药的血脂正常小鼠中血管紧张素II（AngII）诱导的AAA形成， β-氨基丙腈（BAPN）;模拟物介导的miR-146 a过表达消除了AngII诱导的AAAs。 高胆固醇血症LDLr-/-小鼠和正常血脂小鼠均与BAPN共输注。为了阐明 通过RNA测序，我们从miR-146 a缺陷实验中鉴定了新的靶点：TFIID-31， TATA结合蛋白相关因子参与转录激活和抑制， 上调;而Beclin-1，自噬诱导不可或缺的基因和USP 9 X，去泛素化酶 对于Beclin-1稳定至关重要的蛋白质被显著下调。自噬是一种自我调节的过程， 在压力下，细胞消化并回收它们的细胞质材料用于能量目的。我们的初步 一项研究还表明，小鼠AAA中Beclin-1的增加，与人AAA和Tat-肽中观察到的一样 介导的Beclin-1激活抑制小鼠中AngII诱导的AAA形成。此外，miR-146 a 过表达显著抑制TFIID-31，促进USP 9 X和Beclin-1，以及ShRNA介导的 TFIID-31的沉默增加了培养的主动脉SMC中的USP 9 X。根据这些观察，我们将测试我们的 中心假设，miR-146 a激活通过促进AAA形成和进展来保护AAA Beclin-1介导的主动脉SMC稳态。通过利用我们独特的小鼠模型， 在这些研究中，我们提出了三个目标。目的1将检验我们的工作子假设，即miR-146 a促进Beclin-1 通过TFIID-31-USP 9 X依赖性方式在主动脉SMC中稳定。目标2将检验我们的工作子假设 miR-146 a激活通过激活SMC-Beclin-1衍生的自噬来保护免受AAA。目标3 将确定miR-146 a/ Beclin-1激活对已建立的AAA进展的影响。总之，我们 将描述Beclin-1在AAA中的保护作用，并将miR-146 a激活作为一种新的治疗方法。 通过靶向SMC-Beclin-1来对抗AAA的策略。这一机制研究将为临床前研究提供可靠的证据 靶向miR-146 a代表了治疗和预防AAA的新治疗策略。

Abstract Abdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta with a high mortality greater than 80% after rupture. Aortic vascular smooth muscle cells (SMCs) are pivotal in maintaining aortic structural integrity and function, and SMC-rich aortic medial stability is highly disrupted in AAA. Currently, besides surgical interventions, no alternative therapeutics are available to blunt AAA progression and rupture. Consequently, there is a dire need to identify novel strategies for development of effective, non-surgical therapeutics. MicroRNA-146a (miR-146a), a well-known regulator of inflammation and auto-immunity, is highly expressed in aneurysmal tissue of AAA patients. However, the role of miR-146a in SMC homeostasis and AAA remains to be explored. In preliminary studies, by in-situ hybridization, we observed that miR-146a is upregulated in SMC-rich aortic media of human and mouse AAAs; miR-146a deficiency significantly promoted Angiotensin II (AngII) -induced AAA formation in normolipidemic mice co-administered with Lysyl oxidase inhibitor, β-aminopropionitrile (BAPN); and mimetics-mediated miR-146a overexpression abolished AngII-induced AAAs in both hypercholesterolemic LDLr-/- mice and normolipidemic mice co-infused with BAPN. To elucidate underlying mechanisms, by RNA sequencing, we identified novel targets from miR-146a deficiency experiments: TFIID-31, a TATA binding protein associated factor involved in transcriptional activation and repression, is significantly upregulated; whereas Beclin-1, a gene indispensable for autophagy induction and USP9X, a deubiquitinase critical for Beclin-1 stabilization, are significantly downregulated. Autophagy, a self-regulatory process by which cells digest, and recycle their cytoplasmic materials for energy purposes under stress. Our preliminary study also showed an increased Beclin-1 in mouse AAAs, as observed in human AAAs and Tat-peptide mediated Beclin-1 activation suppressed AngII-induced AAA formation in mice. In addition, miR-146a overexpression significantly suppressed TFIID-31, promoted USP9X and Beclin-1, and ShRNA-mediated silencing of TFIID-31 increased USP9X in cultured aortic SMCs. Based on these observations, we will test our central hypothesis that miR-146a activation protects against AAA formation and progression by promoting Beclin-1-mediated aortic SMC homeostasis. By utilizing our unique mice models generated specifically for these studies, we propose 3 aims. Aim 1 will test our working sub-hypothesis that miR-146a promotes Beclin-1 stability in aortic SMCs via a TFIID-31-USP9X –dependent manner. Aim 2 will test our working sub-hypothesis that miR-146a activation protects against AAA through activation of SMC-Beclin-1-derived autophagy. Aim 3 will determine the effect of miR-146a / Beclin-1 activation on progression of established AAAs. In summary, we will delineate the protective role of Beclin-1 in AAA and establish miR-146a activation as a novel therapeutic strategy against AAA by targeting SMC-Beclin-1. This mechanistic research will set solid preclinical evidence that targeting miR-146a represents a novel therapeutic strategy for treatment and prevention of AAA.