The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). We have previously shown that a potential limitation to relaxin-based IPF therapy is decreased expression of a relaxin receptor, relaxin/insulin-like family peptide receptor 1 (RXFP1), in IPF fibroblasts. The mechanism that down-regulates RXFP1 in IPF remains unclear. To determine whether microRNAs (miRs) regulate RXFP1 gene expression, here we employed a bioinformatics approach to identify miRs predicted to target RXFP1 and identified a putative miR-144-3p target site in the RXFP1 mRNA. In situ hybridization of IPF lung biopsies revealed that miR-144-3p is expressed in fibroblastic foci. Furthermore, we found that miR-144-3p is up-regulated in IPF fibroblasts compared with lung fibroblasts from healthy donors. Transforming growth factor β increased miR-144-3p expression in both healthy and IPF lung fibroblasts in a SMAD family 2/3 (SMAD2/3)-dependent manner, and Jun proto-oncogene AP-1 transcription factor subunit (AP-1) was required for constitutive miR-144-3p expression. Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced α-SMA expression. Of note, a lentiviral luciferase reporter carrying the WT 3' UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p-binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates RXFP1 in IPF lung fibroblasts by targeting its 3' UTR. We conclude that miR-144-3p directly represses RXFP1 mRNA and protein expression.

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