Abstract 14698: Matrix Stiffening Induces a Pathogenic Qki-microrna-7-srsf1 Axis in Pulmonary Arterial Endothelial Cells

Abstract

Introduction: Early increases in pulmonary artery stiffness drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling, a prominent feature of pulmonary hypertension (PH). However, the molecular mechanisms by which PAECs respond to altered extracellular matrix (ECM) mechanics remain unclear. Recent RNA sequencing study of PAECs exposed to stiff versus soft matrices has identified miRNA-7 (miR-7) as a mechanosensitive downregulated miRNA. The biogenesis of miR-7 is inhibited by the RNA binding protein quaking (QKI), which is shown to be correspondingly upregulated in ECM stiffness. Moreover, subsequent RNA sequencing and computational analyses have identified serine and arginine rich splicing factor 1 (SRSF1) as a miR-7 target in PAECs with ECM stiffening. Hypothesis: The QKI-miR-7-SRSF1 axis is a pivotal ECM stiffening-mediated pathogenic signal in PAECs. Methods: Human PAECs were cultured on soft (0.5 or 1 kPa) or stiff (50 kPa) matrices. Lungs from PH patients and rats (SU5416/Hypoxia- and MCT-exposed models) were used to evaluate genes and miRNA expression. Boyden chamber migration analysis examined PAEC mobility. Results: In human and rat PH lungs, miRNA-7 (miR-7) was down-regulated ( P < 0.05). To validate RNA sequencing data by RT-qPCR, QKI level was increased (2.13 ± 0.19, P <0.0001) in PAECs cultured on stiff versus soft matrices and correspondingly miR-7 level was decreased (0.71 ± 0.17, P <0.0001). Furthermore, miR-7 targeted the 3’-untranslated region of SRSF1 mRNA as demonstrated by luciferase activity assay. In PAECs, forced miR-7 overexpression decreased SRSF1, and conversely miR-7 inhibition increased SRSF1. QKI overexpression increased SRSF1 by downregulating miR-7 level. Moreover, forced miR-7 overexpression significantly inhibited PAEC migration ( P <0.0001), accompanied by downregulation of SRSF1 mRNA level (0.75 ± 0.07, P <0.0001). Conclusions: Our data demonstrate that mechanoactivation of a QKI-miR-7-SRSF1 signal axis promotes PAEC pathophenotypes driven by ECM stiffness. These insights broaden our understanding of RNA pathobiology in vascular stiffening and carry important therapeutic implications in PH.