MicroRNA in Functional Regulation of Endothelial Cells in Response to Flow

描述（由申请人提供）：MicroRNAs （miRs）是一种小的非编码rna，在调节mRNA稳定性和翻译抑制中起着至关重要的作用。越来越多的证据表明，miRs可以调节心血管系统的基因表达。血管腔内的内皮细胞（ECs）对流体剪切应力等机械因素敏感。在过去的二十年中，该研究小组一直致力于ECs机械转导的机制和随之而来的基因表达。他们和其他人的结果表明，净向前方向的稳定和脉冲剪切应力（PS）通过诱导参与抗增殖和抗炎症的基因来抗动脉粥样硬化。相反，没有明显正向的振荡剪切应力（OS）通过激活促增殖和促炎症基因而促进动脉粥样硬化。基于文献中的新证据和我们最近的发现，miRs在调节EC基因中发挥重要作用，我们假设抗动脉粥样硬化（PS）和促动脉粥样硬化（OS）血流模式诱导不同的miRs模式，从而导致不同的基因表达和功能后果。我们将使用体外、体内和计算机方法来开发一个综合系统，以阐明miRs在不同血流模式下调节EC功能中的作用。这multi-P.I。本研究将结合培养的内皮细胞和小鼠模型的实验数据，结合分子、基因组学和系统方法，阐明miRs在血流作用下内皮细胞功能调控的机制。为了验证我们的假设，我们提出了以下五个具体目标：(1)建立培养的内皮细胞对PS和OS的miR表达谱。(2)确定miRs对PS和OS反应的靶mrna。(3)破译PS与OS下miRs调控的功能基因表达谱。(4)阐明PS和OS下miR调控的功能后果。(5)验证miRs在体内不同血流模式下血管内皮细胞功能调控中的作用。在本文中，我们将结合实验和计算方法，研究miR在不同血流模式下调节血管功能的作用，从miR / mrna到细胞功能进行多尺度分析。这个创新的多学科项目包括(a)全面的全基因组方法来建立ECs中的miR谱，(b) CLIP-seq方法来阐明miRs与靶mrna之间的相互作用，(c)系统生物学方法来绘制miRs调节的功能基因表达和生物学后果，以及(d)在损伤诱导小鼠体内的方法来验证miRs在体外建立的不同流动模式下的作用。该结果将在系统生物学水平上增强对机制调节和功能基因组学作用的机制见解，并可能有助于开发心血管疾病诊断和治疗的新方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in the regulation of mRNA stability and translational repression, leading to the modulation of ~30% of the human genome. We will use in vitro, in vivo, and in silico technologies to develop an integrated systems approach to elucidate the roles of miRs in regulating endothelial functions in normal and pathophysiological flow conditions. The result may and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases.