MicroRNA modulation of smooth muscle cell differentiation

血管平滑肌细胞（vSMCs）支持血管，并为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感兴趣。鉴于在斑马鱼中进行遗传研究的杰出能力，我们已经制定了一个在遗传和细胞水平上研究平滑肌发育的计划。MicroRNAs （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“可调的”，而不是一个简单的开关。这很有趣，因为SMCs是我们身体中为数不多的未最终分化的细胞类型之一，可以将表型从收缩型转换为合成型。利用斑马鱼模型，我们首次在体内研究了mir26的功能，表明mir26的敲低会导致体内出血、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。

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.