Research Paper Volume 16, Issue 19 pp 12909—12927

Figure 6. SOX6 is a target of miR-342-3p. (A) RNA-sequencing analysis showed that SOX6 was an up-regulated differentially expressed gene (fold change ≥ 2.0; p < 0.01) in fibrotic tissues samples (OSF; n=2) compared to normal tissues samples (N; n=2). (B) An illustration of the predicted pairing region between miR-342-3p and SOX6 3’UTR were discovered using the miRDB database, and the 3’ UTR regions of full-length (wt-SOX6) and mutated SOX6 (mut-SOX6) complementarity to the seed site of miR-342-3p, predicted by the TargetScan in silico browser. (C) 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 (B). Luciferase reporter activity was measured 24 hours post-transfection. Data are presented as mean ± SD (n = 3); *p < 0.05 vs. wt-SOX6 with miR-Src. (D) Fibrotic BMFs (−1 and −2) were transfected with either miR-Src or miR-342-3p inhibitor for 24 hours, followed by Western blotting analysis to determine the protein expression of SOX6. (E–H) Normal BMFs (−1 and −2) were transfected with lentiviruses expressing control vector (Ctrl.) or SOX6 (ov-SOX6). The overexpression efficiency of SOX6 was assessed by Western blotting analysis (E). Cells (nBMFs−1 and −2) were cultured in collagen gel for an additional 48 hours, and the resulting gel area after cell contraction was measured (F). Cells (nBMFs−1 and −2) were cultured in Transwell system for an additional 24 hours, and their migration ability was quantified (G). Confluent monolayers of cells (nBMFs−1 and −2) were scratched and cultured for an additional 48 hours, and the wound closure was assessed (H). Data are presented as mean ± SD (n=3); *p < 0.05 vs. Ctrl. (F–H). Scale bar, 50 μm.