Modulation of Endothelial Cell Function by the Shear Stress-Responsive miR-155

描述（由申请人提供）：本提案的目的是确定microRNA表达对内皮细胞功能特异性变化的影响，这些变化是在剪切应力的作用下发生的。血流产生的剪切应力在调节血管张力、血管重塑和动脉粥样硬化病变的局灶发展中起重要作用。在动脉树中，内皮细胞暴露在不同的剪切应力下，对基因表达和功能产生不同的影响。单向剪切应力发生在树的直部分，引起内皮基因表达的变化，通常具有抗炎和动脉粥样硬化保护作用。相反，振荡剪切应力，发生在动脉树的分支点，诱导整体的促炎和促动脉粥样硬化反应。MicroRNAs （miRNAs）是最近发现的一类短（19- 25nt）、单链、非编码rna，已成为分子生物学研究的主要焦点，因为它们转录后调节参与一系列细胞功能的基因的表达，包括分化、生长、增殖和凋亡。尽管已经证明miRNA表达在多种生物过程中发挥重要作用，包括心脏发生和血管生成，但目前关于特定miRNA在内皮细胞生物学中的作用的数据有限。在对长时间单向剪切应力（24小时，15 dynes/cm2）作用的人内皮细胞的初步研究中，发现了一组miRNAs，其表达在这种刺激下显著上调，这表明这些miRNAs在调节内皮细胞的基因表达和功能中起重要作用。为了进一步确定miRNA表达在调节剪切应力诱导的内皮细胞生物学变化中的作用，我们将研究一种高度响应剪切的miRNA miR-155的功能。具体而言，本研究将明确mir -155靶基因相互作用对内皮细胞凋亡、屏障功能和迁移的影响。为了研究miR-155调控细胞凋亡的机制，我们将重点关注SHIP1/PI3K/Akt通路。为了研究miR-155调节内皮单层通透性和迁移的机制，我们将重点研究RhoA/Rho激酶途径。我们将通过实验操纵miR-155、其靶基因或mirna -靶基因相互作用下游通路成员的表达来测试miR-155对这些关键通路的影响。随后，这些操作对内皮细胞凋亡、单层通透性和迁移的影响将被量化。最后，剪切诱导的miR-155表达与内皮细胞调节通路活性之间的关系将在体内进行研究，在主动脉血流改变的小鼠模型中。我们预计这些研究将有助于解决我们关于内皮细胞中mirna功能的知识缺陷，并将增强我们对剪切应力调节血管疾病的机制的理解。

DESCRIPTION (provided by applicant): The purpose of this proposal is to define the influence of microRNA expression on specific changes in endothelial cell function that occur in response to shear stress forces. Shear stress forces, generated by blood flow, play an important role in the regulation of vascular tone, vascular remodeling, and the focal development of atherosclerotic lesions. In the arterial tree, endothelial cells are exposed to different shear stress forces that induce distinct effects on gene expression and function. Unidirectional shear stress, which occurs in the straight part of the tree, elicits a change in endothelial gene expression that is generally anti-inflammatory and atheroprotective. In contrast, oscillatory shear stress, which occurs at branch points in the arterial tree, induces an overall pro-inflammatory and proatherosclerotic response. MicroRNAs (miRNAs) are a recently recognized class of short (19-25 nt), single stranded, noncoding RNAs that have become a major focus in molecular biology research because they posttranscriptional regulate the expression of genes involved in an array of cell functions, including differentiation, growth, proliferation, and apoptosis. Although an important role for miRNA expression has been demonstrated for various biological processes, including cardiogenesis and angiogenesis, data on the role of specific miRNAs in endothelial cell biology is currently limited. In preliminary studies of human endothelial cells subjected to prolonged unidirectional shear stress (24 hrs, 15 dynes/cm2), a group of miRNAs was identified whose expression was significantly upregulated in response to this stimulus, suggesting that these miRNAs are important in regulating gene expression and function in endothelial cells. To further define the role of miRNA expression in modulating shear stress-induced changes in endothelial cell biology, the function of one highly shear-responsive miRNA, miR-155, will be studied. Specifically, the proposed research will define the impact of miR-155-target gene interaction on endothelial cell apoptosis, barrier function and migration. To study the mechanism by which miR-155 modulates apoptosis, we will focus on the SHIP1/PI3K/Akt pathway. To study the mechanism by which miR-155 modulates endothelial monolayer permeability and migration, we will focus on the RhoA/Rho kinase pathway. We will test the influence of miR- 155 on these critical pathways by experimentally manipulating expression of miR-155, its target gene, or members of the pathway that are downstream of the miRNA-target gene interaction. Subsequently, the effect of these manipulations on endothelial cell apoptosis, monolayer permeability, and migration will be quantified. Finally, the association between shear-induced miR-155 expression and activity of endothelial cell regulatory pathways will be studied in vivo, in a mouse model of altered aortic flow. We anticipate that these studies will help address a deficit in our knowledge about the function of miRNAs in endothelial cells and will enhance our understanding of the mechanisms by which shear stress forces modulate vascular disease.