Pathological extracellular matrix suppresses microRNA-29 at the level of microRNA processing in Idiopathic Pulmonary Fibrosis

Abstract

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disorder that kills 40,000 Americans each year. It is characterized by pathological accumulation of activated myofibroblasts and extracellular matrix (ECM) which compromises gas exchange leading to death by asphyxiation. The IPF lung is an order of magnitude stiffer than normal. When primary human lung fibroblasts are cultured on IPF decellularized lung tissue (IPF-ECM), translational repression of ECM genes is disabled due to down-regulation of microRNA-29 (miR-29); however, the molecular mechanism is undefined. Stiffness drives fibroblast-to-myofibroblast differentiation; and stiffness has been shown to mediate microRNA expression. We therefore hypothesized that IPF-ECM decreases miR-29 due to increased matrix stiffness. Methodology: We cultured primary human lung fibroblasts on polyacrylamide hydrogels corresponding to physiological or IPF stiffness, or on Control- or IPF-ECM; and quantified precursor or mature miR-29 (Q-PCR). On hydrogels and decellularized ECM, we defined microRNA processing activity using a luciferase based construct. Results: IPF-ECM caused decreased mature miR-29 and increased precursor miR-29. In contrast, stiffness increased mature miR-29, and decreased precursor miR-29. MicroRNA processing activity was increased by both IPF-ECM and stiffness. Conclusion: On hydrogels simulating pathological stiffness, a stiffness sensor provides a physiological negative feedback signal to increase miR-29, which in turn represses connective tissue synthesis. IPF-ECM mediates decreased miR-29, overriding what should be a negative feedback response to stiffness. Both IPF-ECM and pathological stiffness increases microRNA processing activity, which suggests primary microRNA is converted to precursor microRNA and that IPF-ECM deregulates microRNA processing at the level of post-microprocessor complex. Thus, our data is the first to identify corruption of microRNA processing by the IPF fibrotic matrix itself as one mechanism for sustained connective tissue production in IPF.