MicroRNA modulation of smooth muscle cell differentiation

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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.