Figure 9.

miR-144-3p directly targets the 3′ UTR of RXFP1 mRNA. A, depiction of pmirGLO dual-luciferase reporter construct for the WT 3′ UTR of the RXFP1 seed region and mutations. B and C, 293T cells were plated 24 h prior to transfection. Cells were then co-transfected with 50 ng of either pmirGLO vector carrying WT (B) 3′ UTR of RXFP1 or mutated (C) 3′ UTR of RXFP1 with and without 100 nm miR-144-3p mimic or control mimic. miR-144-3p significantly repressed the luciferase activity of the reporter containing the WT 3′ UTR of RXFP1 (p < 0.005, unpaired t test, mean ± S.D., n = 3), whereas the mutated construct was insensitive in 293T cells (I, unpaired t test, mean ± S.D., n = 3). Luciferase activity was measured using Renilla luciferase as an internal control. D, lentiviral luciferase reporter construct carrying WT 3′ UTR of RXFP1 was used to transduce both donor and IPF lung fibroblasts. There was a reduction in luciferase activity with WT 3′ UTR of RXFP1 compared with mutated 3′ UTR of RXFP1. Also, the luciferase activity of WT 3′ UTR RXFP1 was highly suppressed in IPF lung fibroblasts compared with donor lung fibroblasts indicating higher levels of endogenous miR-144-3p in IPF lung fibroblasts compared with that of donor. Lentiviral Renilla luciferase vector was used as an internal control. Data shown are from a representative experiment (n = 3). E, depiction of mmu–miR-144-3p mimic and its modified version to enhance base pairing with mouse RXFP1 3′ UTR. There is a single nucleotide mismatch in mmu–miR-144-3p with mouse RXFP1 3′ UTR target region. E, primary MLF were isolated from C57/B6 mice (n = 2; repeated twice). MLF were transfected either with a mmu–miR-144-3p mimic or a modified mmus–miR-144-3p mimic (modified to match mouse RXFP1 target region) along with a negative control mimic. MLF were harvested to isolate total RNA and qRT-PCR was performed to determine the levels of mRXFP1, mPPIA, and mNFE2L2 (mNrf2).