RXFP1 expression is regulated by miR-144-3p in Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis

Abstract

Relaxin has been considered as a potential therapy for patients with pulmonary fibrosis. We have previously shown, however, that a potential limitation of relaxin-based therapy for Idiopathic Pulmonary Fibrosis (IPF) is the loss of expression of the relaxin receptor Relaxin/Insulin Like Receptor 1 (RXFP1) expression in fibroblasts. The molecular mechanism for RXFP1 down-regulation in IPF patients remains unclear. To determine whether microRNAs play a role in RXFP1 gene expression, we employed a bioinformatics approach to identify microRNAs (miRs) that are predicted to target RXFP1. By in silico analysis, we identified a putative target site in the RXFP1 mRNA for the miR-144 family. We found that miR-144-3p was upregulated in IPF fibroblasts compared to donor lung fibroblast controls. Forced miR-144-3p mimic expression reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker alpha-smooth muscle actin (a-SMA) in donor lung fibroblasts. IPF lung fibroblasts transfected with a miR-144-3p inhibitor increased RXFP1 expression and reduced a-SMA expression. A lentiviral luciferase reporter vector carrying the WT 3'UTR of RXFP1 was repressed more in lung fibroblasts whereas vector carrying a mutated miR-144-3p binding site exhibited less sensitivity to endogenous miR-144-3p expression, Conflict of interest: Authors declare there is no conflict of interest. Author Contributions: JT, HB, JAD, and SBS conducted experiments and analyzed the results. JT, JAD, SBS conducted qRT-PCR and western blot experiments. HB conducted the luciferase experiments. DJK, JT, and YZ conceived and planned experiments. HB, JT, JAD, SBS, YZ and DJK wrote the manuscript.