MicroRNA modulation of smooth muscle cell differentiation

血管平滑肌细胞(VSMCs)支持血管，对其收缩和稳定出血的能力是不可或缺的。我们对血管平滑肌分化的分子调控很感兴趣。鉴于斑马鱼出色的遗传研究能力，我们开发了一个在遗传和细胞水平上研究平滑肌肉发育的项目。MicroRNAs(MiRs)是一种短的RNA序列，通过靶向翻译抑制或降解的mRNA来控制基因的表达。MIR对基因表达的控制是快速可逆或可调的，而不是简单的开关开关。这很有趣，因为SMC是我们身体中为数不多的未终末分化的细胞类型之一，可以将表型从收缩转换为合成。利用斑马鱼模型，我们首次对mir26的功能进行了体内研究，结果表明，下调mir26会导致体内出血、血管内皮细胞与平滑肌细胞接触不良以及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靶点由mir26平行控制，导致平滑肌细胞分化和血管稳定性假设1：mir26通过Smad1控制转化生长因子？信号来控制SMC的分化。在这个目标中，我们首先测试已知的mir26靶点在vSMC发育中的作用。我们将使用体外和体内实验来验证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水平升高对SMC分化的形态影响。这一目标描述了一种试验性的“流水线”战略，可以用来表征更多的目标。假设2：miR26针对的是多个对SMC分化调控至关重要的RNA。在这个目标中，我们从PAR-Clip测序中挖掘已发表的数据集，这是一项对mRNAs进行测序的技术，这些mRNAs是与识别靶点相关的microRNA。从这项技术在癌细胞系中产生的mir26靶点列表中，我们将使用表达筛选来识别在斑马鱼体内SMC分化过程中可能受mir26调控的其他基因。然后将使用目标1中概述的实验策略来研究这些靶点。我们的前两个靶点包括多肽激素肾上腺髓质素和转化生长因子途径成员SKI。假设3：miR26控制的基因将并行工作以促进SMC分化，目标1和2将识别由miR26调控的多个基因，我们假设一些基因将在遗传途径中发挥作用，而另一些基因将并行作用。在这一目标中，我们将利用经典的途径分析来探索miR26调控的遗传网络。假设4：将miR26体内导入SMC将调节它们的遗传程序我们的最终目标是了解miR26是否可用于治疗。由于SMC是直接与内皮衬里相邻的薄细胞，我们假设通过血管造影术在循环中给予MIR将导致MIR被吸收到SMC中。我们将确定标记的miR26的定位和摄取，以及它是否对SMC的发育具有表型效应。意义：我们提出的计划将在活体内识别一组SMC中的直接MIR26靶点。以核酸为基础的药物在理论上是廉价、稳定和易于管理的。因此，抑制或增强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，我们正在通过确定miR26的目标来为这一领域做出贡献。

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.