Research Paper Volume 16, Issue 19 pp 12909—12927

Figure 4. MiR-342-3p negatively correlates to MIAT expression in OSF tissues and acts as an anti-fibrotic miRNA in OSF. (A) RNA of cytoplasmic and nuclear fractions from primary fBMFs (−1 and −2) were analyzed by qRT-PCR to determine the subcellular localization of MIAT. Data are presented as mean ± SD (n=3); *p < 0.05 vs. Nucleus. (B) RNA-sequencing analysis showed that miR-342-3p was a down-regulated differentially expressed gene (fold change ≤ -2.0; p < 0.01) in samples of fibrotic tissues (OSF; n=2) compared to normal tissues (N; n=2). (C) An illustration of the predicted pairing region between miR-342-3p and MIAT 3’UTR, discovered using the miRanda database, and the 3’ UTR regions of full-length (wt-MIAT) and mutated MIAT (mut-MIAT) complementarity to the seed site of miR-342-3p, predicted by TargetScan in silico browser. (D) The relative expression of miR-342-3p in normal (N; n=25) and fibrotic (OSF; n=25) tissues was assessed by qRT-PCR analysis. Data are presented as mean ± SD. (E) A significant negative correlation was observed between MIAT and miR-342-3p expression fibrotic tissue samples (OSF; n=45). (F) Fibrotic BMFs (−1) were co-transfected with either miR-Scramble (miR-Src.) or miR-342-3p mimics, along with the indicated pmirGLO-based constructs shown in (C). Luciferase reporter activity was measured 24 hours post-transfection. Data are presented as mean ± SD (n = 3); *p < 0.05 vs. wt-MIAT with miR-Src. (G–I) Fibrotic BMFs (−1 and −2) expressing Sh-Luc or Sh-MIAT were transfected with either miR-Src or miR-342-3p inhibitor for 24 hours. The cells (fBMFs−1 and −2) were cultured in collagen gel for an additional 48 hours, and the resulting gel area after cell contraction was measured (G). Cells (fBMFs−1 and −2) were cultured in Transwell system for an additional 24 hours, and their migration ability was quantified (H). Data are presented as mean ± SD (n=3); *p < 0.05 vs. miR-Scr. (G, H). Confluent monolayers of fBMF−2 were scratched and cultured for an additional 48 hours, and the wound closure was assessed (I). Scale bar, 50 μm.