MicroRNA in Functional Regulation of Endothelial Cells in Response to Flow

项目摘要： 微小RNA（miRs）是小的非编码RNA，其在调节mRNA稳定性中起关键作用， 翻译抑制越来越多的证据表明，miR可以调节肿瘤细胞中的基因表达。 心血管系统血管内皮细胞对机械力非常敏感 例如流体剪切应力。在过去的二十年里，这个研究小组一直致力于研究 EC中的机械转导和随后的基因表达。他们和其他人的结果表明， 具有净向前方向的稳定和脉动剪切应力（PS）通过诱导 抗增殖和抗炎基因。相比之下，振荡剪切应力（OS）没有 重要的前向方向是通过激活促增殖和促炎基因而促动脉粥样硬化。 基于文献中的新证据和我们最近的发现，miR在调节 EC基因，我们假设抗动脉粥样硬化（PS）和促动脉粥样硬化（OS）的血流模式诱导不同的 miR的模式，因此差异基因表达和功能后果。我们将在 体外、体内和计算机模拟方法来开发一个整合系统，以阐明miR在以下方面的作用： 调节EC功能以响应不同的流动模式。这个多私家侦探研究项目，结合 从培养的EC和小鼠模型中获得的实验数据， 的方法，将阐明的功能调节的机制，在EC的miR下流。为了测试 我们的假设，我们提出了以下五个具体目标：（1）建立miR表达谱在培养的 （2）确定响应于PS vs. OS的miR的靶mRNA。（3）确定响应于PS vs. OS的miR的靶mRNA。 破译PS与OS下miR调控的功能基因表达谱。（4）为了阐明 PS与OS下miR调节的功能后果。（5）为了验证miR在功能性调节中的作用， 调节暴露于体内不同流动模式的血管EC。在这项提议中，miR在 调节血管功能将在不同的流动模式下进行研究， 和计算方法来执行从miR/mRNA到细胞功能的多尺度分析。这 一个创新的、多学科的项目包括：（a）建立miR的全基因组方法 （B）阐明miR和靶mRNA之间相互作用的CLIP-seq方法，（c） 系统生物学方法来映射功能基因表达和生物学后果的调控 miR，和（d）在损伤诱导小鼠中的体内方法，以验证miR在不同流动条件下的作用 在体外建立的模式。研究结果将有助于从机理上理解机械-机械- 调控和功能基因组学在系统生物学水平，并可能有助于发展 诊断和治疗心血管疾病的新方法。

Project Summary: MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in regulating mRNA stability and translational repression. There is increasing evidence that miRs can modulate gene expression in the cardiovascular system. Endothelial cells (ECs) lining the vascular lumen are sensitive to mechanical factors such as fluid shear stress. During the past two decades, this research team has worked on the mechanisms of mechanotransduction in ECs and the consequent gene expression. The results from them and others indicate that steady and pulsatile shear stresses (PS) with a net forward direction are anti-atherogenic by inducing genes involved in anti-proliferation and anti-inflammation. In contrast, oscillatory shear stress (OS) without a significant forward direction is pro-atherogenic by activating pro-proiferative and pro-inflammatory genes. Based on new evidence in the literature and our recent findings that miRs play an important role in regulating EC genes, we hypothesize that anti-atherogenic (PS) and pro-atherogenic (OS) flow patterns induce distinct patterns of miRs, and hence the differential gene expressions and functional consequences. We will use in vitro, in vivo, and in silico approaches to develop an integrated system to elucidate the roles of miRs in regulating EC functions in response to different flow patterns. This multi-P.I. research project, by combining experimental data obtained from cultured ECs and mouse models with molecular, genomics and systems approaches, will elucidate the mechanisms of functional regulation by miRs in ECs under flows. In order to test our hypothesis, we propose the following five specific aims: (1) To establish miR expression profiles in cultured ECs in response to PS vs. OS. (2) To determine the target mRNAs of miRs in response to PS vs. OS. (3) To decipher the functional gene expression profiles regulated by miRs under PS vs. OS. (4) To elucidate the functional consequences of miR regulation under PS vs. OS. (5) To verify the role of miRs in functional regulation of vascular ECs exposed to different flow patterns in vivo. In this proposal the role of miR in regulating vascular functions will be studied under different flow patterns with a combination of experimental and computational approaches to perform multi-scale analyses from miRs/mRNAs to cellular functions. This innovative, multidisciplinary project includes (a) comprehensive genome-wide approaches to establish the miR profiles in ECs, (b) CLIP-seq approaches to elucidate the interactions between miRs and target mRNAs, (c) systems biology approaches to map the functional gene expression and biological consequence regulated by miRs, and (d) in vivo approaches in lesion-induction mice to validate the roles of miRs under different flow patterns established in vitro. The results will enhance the mechanistic insights of the roles of mechano- regulation and functional genomics at the systems biology level and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases.