Pneumologie 2015; 69 - A42
DOI: 10.1055/s-0035-1556634

MicroRNA-9 is a regulator of PDGFRβ expression in pulmonary arterial hypertension

A Weiss 1, K Gernert 1, S Schaefer 1, L Maegel 2, D Jonigk 2, A Ghofrani 1, N Weissmann 1, F Grimminger 1, W Seeger 1, R Schermuly 1
  • 1UGMLC, Gießen
  • 2MHH, Hannover

Introduction:

In several diseases, proteins are differentially expressed compared to healthy conditions due to microRNAs which serve as translational repressors. Therefore, we aim to define the role of microRNAs in the de-regulation of PDGFRβ expression and their impact on the onset and progression of PAH.

Methods:

In this project, a variety of techniques commonly used in molecular biology were applied. Western blot analysis and realtime PCR were performed for expression profiling of proteins and mRNAs/microRNAs in cell extracts from lung tissue of mice and humans. The hypoxia-induced mouse model was used to resemble the pathological phenotype of structural remodeling in the lung vasculature during PAH. Human material from healthy individuals and IPAH patient was kindly provided by the UGLC Biobank. Cell culture experiments with commercially available primary human pulmonary artery smooth muscle cells were conducted including transfection of microRNA mimics and inhibitors. Cloning of plasmids and subsequent luciferase reporter assays according to standard protocols allowed the investigation of microRNA binding to the mRNA of the PDGFRβ.

Results:

We identified miR-9 amongst other microRNAs as a potential candidate involved in translational repression of PDGFRβ mRNA. Our expression analyses confirmed the over-activation of PDGFRβ and demonstrated its inverse correlation to the endogenous miR-9 expression in the lung homogenates of hypoxia-treated mice. We also evaluated the binding of miR-9 to the 3'UTR of the PDGFRβ mRNA followed by the inhibition of translation into PDGFRβ protein. Next, we investigate the effects of a re-constitution of normal miR-9 levels on the PDGFRβ expression in primary vascular cells (as well as in vivo). Here, we focus also on the cellular processes of structural remodeling namely proliferation, survival and migration.

Discussion:

In summary, this project allows us to understand the role of microRNAs, particularly miR-9, as negative regulators of PDGFRβ expression in this life-threatening disease.

*Presenting author