Novel role of the lncRNA NEAT1 in smooth muscle phenotypic modulation

 描述（由申请人提供）：拟议研究的总体目标是确定（长链非编码RNA）lncRNA NEAT 1（核旁斑组装转录本1）先前未被认识到的作用及其在调节血管平滑肌细胞（VSMC）表型转换中的新潜在机制。人类的许多血管疾病，如血管成形术后内膜增生，在很大程度上依赖于VSMC从收缩表型转换为“合成”表型，这与平滑肌特异性基因表达减少和细胞增殖和迁移增加有关。因此，阐明VSMC表型转换的机制对于理解VSMC相关血管疾病的病理学和最终设计用于治疗的治疗剂至关重要。越来越多的证据表明，lncRNA代表了一类新的基因表达调控因子。为了寻找参与血管损伤的lncRNA，我们使用球囊剥脱损伤的大鼠颈动脉进行了大规模lncRNA阵列筛选，球囊剥脱损伤是一种类似于人类血管成形术的手术。该筛选揭示lncRNA NEAT 1在体内响应动脉损伤而被诱导。此外，在体外刺激平滑肌表型调节时也诱导NEAT 1的表达。NEAT 1功能的丧失和获得表明，NEAT 1不仅增加VSMC的增殖和迁移，而且降低平滑肌特异性收缩蛋白的表达。因此，本提案中描述的实验将检验NEAT 1的诱导在促进平滑肌表型转换中起关键作用的假设。在目标1中，我们将确定NEAT 1在动脉损伤后新生内膜增生中的作用。我们将在对照和NEAT 1敲除小鼠中进行股动脉钢丝损伤，并评估NEAT 1 KO对新生内膜增生进展的影响。在目标2中，我们将确定NEAT 1消除平滑肌特异性基因表达的机制。具体来说，我们将评估WDR 5的作用，一个关键的组蛋白修饰剂诱导染色质的活性形式，在介导NEAT 1功能的VSMC。在目标3中，我们将通过测试NEAT 1近端启动子中鉴定的进化保守的cAMP反应元件的作用，确定NEAT 1转录在刺激平滑肌表型调节后上调的机制。这些研究的完成将为控制平滑肌表型转换的机制提供新的见解，并确定防止NEAT 1的诱导是否可能是改善闭塞性血管疾病的有吸引力的治疗策略。

 DESCRIPTION (provided by applicant): The overall goal of the proposed research is to determine the previously unrecognized role of the (long non-coding RNA) lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1) and its novel underlying mechanism in regulating the phenotypic switching of vascular smooth muscle cells (VSMCs). Many vascular diseases in humans, such as intimal hyperplasia post-angioplasty, are largely dependent upon VSMC phenotypic switching from a contractile to a "synthetic" phenotype that is associated with reduced smooth muscle-specific gene expression and increased cell proliferation and migration. Therefore, unraveling the mechanisms for the VSMC phenotypic switching is crucial for understanding the pathology of VSMC related vascular diseases and ultimately designing therapeutic agents for treatment. Emerging evidences demonstrate that lncRNAs represent a novel class of regulators for gene expression. In an effort to search for lncRNAs involved in the vascular injury, we conducted a large-scale lncRNA array screen using the rat carotid artery injured by a balloon denudation, a procedure resembling angioplasty in humans. This screen revealed that the lncRNA NEAT1 is induced in response to the arterial injury in vivo. Furthermore, expression of NEAT1 is also induced upon the stimulation of smooth muscle phenotypic modulation in vitro. Loss- and gain-of-function NEAT1 revealed that NEAT1 not only increases proliferation and migration of VSMCs but also decreases expression of smooth muscle-specific contractile proteins. Therefore, experiments described in this proposal will test the hypothesis that, the induction of NEAT1 plays a critical role in promoting smooth muscle phenotypic switching. In Aim 1, we will determine the role of NEAT1 in neointima hyperplasia following arterial injury. We will perform femoral artery wire injuries in the control and NEAT1 knock out mice and assess the effects of NEAT1 KO on the progression of neointima hyperplasia. In Aim 2, we will determine the mechanism by which NEAT1 abrogates smooth muscle-specific gene expression. Specifically we will evaluate the role of WDR5, a critical histone modifier for inducing an active form of chromatin, in mediating NEAT1 function in VSMCs. In Aim 3, we will determine the mechanism by which the transcription of NEAT1 is up-regulated upon the stimulation of smooth muscle phenotypic modulation by testing the role of an evolutionally conserved cAMP response element identified in the NEAT1 proximal promoter. Completion of these studies will provide novel insights into the mechanisms controlling smooth muscle phenotypic switching and determine if preventing the induction of NEAT1 may be an attractive therapeutic strategy for ameliorating occlusive vascular diseases.