MicroRNA in Functional Regulation of Endothelial Cells in Response to Flow

描述(申请人提供)：microRNAs(MiRs)是一种小的非编码RNA，在调节mRNA稳定性和翻译抑制方面发挥关键作用。越来越多的证据表明，miRs可以调节心血管系统中的基因表达。血管腔内的内皮细胞(ECs)对流体剪应力等机械因素非常敏感。在过去的二十年里，这个研究小组致力于研究内皮细胞的机械转导机制和随后的基因表达。他们和其他人的结果表明，稳定和脉动的切应力(PS)具有净正向，通过诱导参与抗增殖和抗炎的基因来抗动脉粥样硬化。相反，没有显著正向的振荡切应力(OS)通过激活促增殖和促炎基因而促进动脉粥样硬化的形成。根据文献中的新证据和我们最近发现的MIR在EC基因调控中的重要作用，我们假设抗动脉粥样硬化(PS)和促进动脉粥样硬化(OS)的流动模式会导致不同的MIR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、体内和硅胶方法开发一个集成的系统，以阐明MIR在调节EC功能中的作用，以响应不同的流动模式。通过将培养的内皮细胞和小鼠模型获得的实验数据与分子、基因组学和系统学方法相结合，这一多PI研究项目将阐明MIR在血管内皮细胞中的功能调节机制。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PS与OS反应的miRs的靶mRNAs。(3)破译PS与OS条件下miRs调控的功能基因表达谱。(4)阐明PS与OS下miR调控的功能后果。(5)验证MIR在体内不同流型下血管内皮细胞功能调节中的作用。在这项提案中，将研究不同流动模式下miR在调节血管功能中的作用，采用实验和计算相结合的方法进行从miRs/mRNAs到细胞功能的多尺度分析。这一创新的多学科项目包括：(A)建立ECs中miR图谱的全面基因组方法，(B)阐明miRs与靶mRNAs之间相互作用的CLIP-SEQ方法，(C)绘制miRs调控的功能基因表达和生物学后果的系统生物学方法，以及(D)在病变诱导小鼠中验证miRs在体外建立的不同流动模式下的作用的体内方法。这些结果将在系统生物学水平上加强对机械调节和功能基因组作用的机械洞察，并可能有助于开发新的心血管疾病诊断和治疗方法。

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.