Global analysis of mRNA polarization in migrating endothelial cells

描述(申请人提供)：血管暴露在多种压力下，可导致内皮损伤，必须进行快速修复才能恢复血管和整个心血管系统的正常功能。内皮细胞(EC)的迁移在这一修复过程中以及在新血管的形成中起着关键作用。多个细胞系统的极化，包括信号、黏附蛋白、质膜、分泌过程、细胞骨架排列等，是诱导和控制细胞迁移的关键。最近的研究表明，2-肌动蛋白mRNA和其他几个与运动相关的转录本聚集在运动细胞的前端，但对极化的mRNAs及其共同的定位元件的总体情况知之甚少。我们的长期目标是了解在EC运动过程中mRNA极化在不对称性发展中的作用，以及平面运动细胞中mRNAs极化的机制。为了实现这一目标，我们启动了对在EC迁移过程中变得两极分化的mRNAs的全面、全球分析。我们应用激光显微解剖方法从平面运动的EC的前部和后部区域捕捉细胞碎片，这是一个代表伤口愈合过程的模型。我们建议将这种微切割方法与微阵列技术相结合，然后进行严格的生化研究，以解决以下假设：mRNAs中的顺式元件决定其在平面迁移EC的正面和背面的定位，并负责将蛋白质靶向其功能部位。我们将通过追求以下特定目标来验证这一假设：(1)寻找在迁移EC中极化的新的mRNAs；(2)研究与细胞迁移过程中的mRNA极化相关的RNA元件。我们的研究将首次对细胞迁移过程中的mRNA定位进行全球分析，并将开始填补我们对迁移过程中细胞极化的理解的重要空白。特别是，人们对诱导mRNAs在细胞前端积累的RNA元件知之甚少，对积累在细胞后部的mRNAs(及其功能元件)几乎一无所知。除了对我们理解细胞极化的机制贡献外，我们的工作还可以揭示对细胞运动调节的新见解，并具有改变运动的新干预措施的潜力。例如，识别新的定位元件为通过基于RNA的方法调节mRNA定位和细胞迁移提供了新的治疗靶点，例如，通过靶向特定RNA元件的反义RNA寡聚体治疗。最后，我们的工作可能会提供并验证一项技术进步，这将允许在其他系统中对不对称mRNA进行全球分析。与公共健康相关：血管暴露在多种压力下，可损伤血管壁的内皮细胞(EC)衬里。内皮细胞随后的迁移对于血管壁修复和正常血管功能的恢复至关重要。编码运动相关蛋白的信使RNA(MRNA)的极化是一个新发现的事件，可能有助于EC运动所需的细胞方向性。我们的研究将首次对平面细胞迁移过程中的mRNA极化进行全球分析，并可能为调控运动相关mRNAs的定位和细胞迁移提供新的治疗靶点。

DESCRIPTION (provided by applicant): Blood vessels are exposed to multiple stresses that can induce injury to the endothelium, which must undergo rapid repair to restore normal function of the vessel and the entire cardiovascular system. Endothelial cell (EC) migration has a critical role in this repair process, as well as in formation of new blood vessels. Polarization of multiple cell systems, including signaling, adhesion proteins, plasma membrane, secretion processes, cyto- skeletal arrangement, etc., is crucial for induction and control of cell migration. Recent studies have shown that 2-actin mRNA, and several other motility-related transcripts, accumulated in the front of moving cells, but little is known about the total ensemble of polarized mRNAs and their common localization elements. Our long-term goal is to understand the contribution of mRNA polarization to the development of asymmetry during EC movement, and the mechanisms by which mRNAs are polarized in planar moving cells. To accomplish this goal, we have initiated a comprehensive, global analysis of the mRNAs that become polarized during EC migration. We have applied the laser microdissection methodology to capture cell fragments from forward and rearward regions of planar-moving EC, a model representing the wound-healing process. We propose to couple this microdissection method with microarray technologies, followed by rigorous biochemical investigation to address the following hypothesis: cis-elements in mRNAs determine their localization in both the front and back of planar-migrating EC, and are responsible for targeting proteins to their site of function. We will test this hypothesis by pursuing the following specific aims: (1) identify novel mRNAs polarized in migrating EC, and (2) investigate RNA elements responsible for mRNA polarization during cell migration. Our studies will provide the first global analysis of mRNA localization during cell migration, and it will begin to fill important gaps in our understanding of cell polarization during migration. In particular, little is known about the RNA elements that induce mRNAs to accumulate in the cell front and virtually nothing is known about mRNAs (and their functional elements) that accumulate in the cell rear. In addition to the mechanistic contribution to our understanding of cell polarization, our work can reveal new insights into the regulation of cell movement, and has the potential of new interventions to alter motility. For example, identification of novel localization elements presents novel therapeutic targets for modulating mRNA localization and cell migration by RNA-based approaches, for example, by treatment with antisense RNA oligomers targeting the specific RNA element. Finally, our work is likely to provide and validate a technological advance that will permit global analysis of asymmetric mRNA in other systems. PUBLIC HEALTH RELEVANCE: Blood vessels are exposed to multiple stresses that can injure the endothelial cell (EC) lining of the vessel wall. Subsequent migration of EC is critical for vessel wall repair and restoration of normal blood vessel function. Polarization of the messenger RNAs (mRNA) encoding movement-related proteins is a newly discovered event that may contribute to cell directionality required for EC movement. Our studies will provide the first global analysis of mRNA polarization during planar cell migration, and it may reveal novel therapeutic targets for modulating localization of motility-related mRNAs and cell migration.