Figure 2.

miR-144-3p targets RXFP1 in human lung fibroblasts. A, the seed region of hsa–miR-144-3p predicted to target the 3′ UTR of human RXFP1. B and C, donor and IPF lung fibroblasts were treated with miR-144-3p mimic or scrambled control. RNA was isolated and processed for qPCR for RXFP1 (B) or RXFP2 (C). Significantly higher basal levels of RXFP1 mRNA were detected in donor lung fibroblasts compared with IPF lung fibroblasts (p = 0.007, n = 3). Treatment of donor lung fibroblasts with the miR-144-3p significantly decreased RXFP1 expression (p = 0.0005, by two-way ANOVA and Tukey post hoc testing, n = 3). D, donor and IPF lung fibroblasts were transfected with 10 nm miR-144-3p mimic or control, and cells were lysed for immunoblotting for RXFP1, α-SMA, and β-actin. E, densitometry of immunoblotting presented in F. Data are expressed as mean ± S.D., normalized to β-actin. Less RXFP1 protein was detected in donor lung fibroblasts following transfection of miR-144-3p compared with scrambled control (p = 0.0016, n = 3). Significantly more RXFP1 was present in donor lung fibroblasts compared with IPF lung fibroblasts following transfection with the scrambled control (p = 0.0019, n = 3). Data were analyzed by two-way ANOVA and Tukey's post-hoc test. F, densitometry of α-SMA were normalized to β-actin. Transfection of miR-144-3p into donor lung fibroblasts significantly increased expression of α-SMA (p = 0.0117 for log-transformed data, n = 3, by two-way ANOVA followed by Tukey's post hoc testing).