MicroRNA modulation of smooth muscle cell differentiation

血管平滑肌细胞（vSMC）支持血管，并且对于它们收缩和稳定出血的能力是不可或缺的。我们感兴趣的是血管平滑肌分化的分子调控。鉴于在斑马鱼中进行遗传研究的杰出能力，我们已经开发了一个在遗传和细胞水平上研究平滑肌发育的计划。微小RNA（miRs）是通过靶向mRNA进行翻译抑制或降解来控制基因表达的短RNA序列。miR对基因表达的控制是快速可逆的或"可调节的"，而不是简单的开关。这很有趣，因为SMC是我们体内少数几种未终末分化的细胞类型之一，可以将表型从收缩型转换为合成型。使用斑马鱼模型，我们进行了mir26功能的第一次体内研究，以表明mir26的敲低导致体内出血、平滑肌-内皮细胞接触不良和SMC分化缺陷。我们提出的研究计划的目标是了解血管是如何稳定的，重点是mir26及其下游靶点。我们的一般假设是，一小群关键的mRNA靶点平行控制的mir26导致平滑肌细胞分化和血管稳定性假设1：mir26控制SMC分化通过控制TGF β 1信号通过smad1在这个目标中，我们将首先测试一个已知的mir26目标，其在vSMC的发展中的作用。我们将使用体外和体内试验来验证smad1是miR26的直接靶点。我们还将使用显微镜和分子标记来确定胚胎发生过程中smad1升高对SMC分化的形态学影响。这个目标描述了一个实验性的“管道”策略，可用于表征其他目标。假设二：miR 26靶向调节SMC分化的多种重要RNA。为此，我们对来自PAR-CLIP测序的已发表数据集进行数据挖掘，PAR-CLIP测序是一种对与鉴定靶标相关的microRNA的mRNA进行测序的技术。从列表中的mir26目标产生的这种技术在癌细胞系中，我们将使用表达筛选，以确定额外的基因可能调控的mir26在体内的平滑肌细胞分化在斑马鱼。然后将使用目标1中概述的实验策略研究这些目标。我们的前两个目标包括肽激素肾上腺髓质素和TGF β通路成员ski。假设三：由miR26控制的基因将并行工作以促进SMC分化。目的1和2将鉴定由miR26调控的多个基因，并且我们假设一些基因将在遗传途径中起作用，而一些基因将并行起作用。在这个目标中，我们将探索miR26调控的遗传网络，使用经典的途径分析。假设4：将miR26体内递送至SMC将调节其遗传程序。我们的最终目标是了解miR26是否可以用于治疗。由于SMC是直接邻近内皮衬里的薄细胞，我们假设通过血管造影术在循环中施用miR将导致miR被SMC吸收。我们将确定标记的miR 26的定位和摄取，以及它是否对SMC发育具有表型效应。意义：我们提出的计划将在体内SMC中鉴定一组直接的mir26靶点。基于核酸的药物在理论上是便宜的、稳定的并且易于施用。因此，抑制或增加miR水平可能是一种可行的治疗策略。为了评估潜在的副作用，我们通过鉴定miR26的靶点来为该领域做出贡献。

Vascular smooth muscle cells (vSMCs) support blood vessels, and are integral for their ability to contract and be stabilized from hemorrhage. We are interested in the molecular control of differentiation of vascular smooth muscle. Given the outstanding ability to undertake genetic studies in zebrafish, we have developed a program to study smooth muscle development at the genetic and cellular levels. MicroRNAs (miRs) are short RNA sequences that control gene expression by targeting mRNA for translational repression or degradation. miR control of gene expression is rapidly reversible or ‘tunable’, rather than a simple on-off switch. This is interesting, as SMCs are one of the few cell types in our body that are not terminally differentiated and can switch phenotypes from being contractile to synthetic. Using the zebrafish model we have taken the first in vivo studies of mir26 function to show that knockdown of mir26 results in hemorrhage in vivo, poor smooth muscle-endothelial cell contacts, and SMC differentiation defects. The goal of our proposed research program is to understand how blood vessels are stabilized with the focus on mir26 and its downstream targets. Our general hypothesis is that a small group of critical mRNA targets are controlled in parallel by mir26 leading to smooth muscle cell differentiation and vascular stability Hypothesis 1: mir26 controls SMC differentiation by controlling TGFß signalling via smad1 In this aim we will first test a known mir26 target for its role in vSMC development. We will use in vitro and in vivo assays to validate that smad1 is a direct target of miR26. We will also determine the morphological consequences of elevated smad1 during embryogenesis on SMC differentiation using microscopy and molecular markers. This aim describes an experimental 'pipeline' strategy that can be used to characterize additional targets. Hypothesis 2: Multiple RNAs important for modulation of SMC differentiation are targeted by miR26 In this aim we data-mine published datasets from PAR-CLIP sequencing, a technique that sequences mRNAs that are microRNA associated to identify targets. From a list of mir26 targets generated by this technique in cancer cell lines, we will use expression screening to identify additional genes potentially regulated by mir26 during SMC differentiation in vivo in zebrafish. These targets will then be studied using the experimental strategy outlined in Aim 1. Our first two targets include the peptide hormone adrenomedullin and the TGFß pathway member ski. Hypothesis 3: Genes controlled by miR26 will work in parallel to promote SMC differentiation Aims 1 and 2 will identify multiple genes regulated by miR26, and we hypothesize that some will act in genetic pathways, while some will act in parallel. In this aim we will explore the genetic network regulated by miR26 using classical pathway analysis. Hypothesis 4: Delivery of miR26 in vivo to SMCs will modulate their genetic program Our ultimate goal is to understand whether miR26 can be used therapeutically. Since SMCs are thin cells directly adjacent to the endothelial lining, we hypothesize that administration of a miR in the circulation by angiography will result in the miR being taken up into SMCs. We will identify the localization and uptake of labelled miR26 and whether it has a phenotypic effect on SMC development. Significance: Our proposed program will identify a group of direct mir26 targets in SMCs in vivo. Nucleic acid based pharmaceuticals are theoretically inexpensive, stable and easy to administer. Therefore inhibition or augmentation of miR levels could be a feasible therapeutic strategy. In order to assess potential side-effects we are contributing to this field by identifying targets of miR26.