Figure 6. Syndecan-1 controls packaging of antifibrotic miRNAs into EVs.

WT and Sdc1^–/– mice were injured with bleomycin (0.75 unit/kg) and sacrificed 21 days after injury. Fibrotic EVs were collected from the airspaces, and miRNAs were isolated for RNA-Seq. (A) Analysis of the miRNA levels within fibrotic EVs from bleomycin-injured WT and Sdc1^–/– mice identified significant differences in 5 miRNAs. (B) Predicted miR-34b-5p binding site in the Muc5b 3′ UTR region was identified with http://www.microrna.org/microrna/home.do (C) Muc5b dot blot of MLE-12 cells transfected with control and miR-34b-5p mimics. (D) Luciferase activity of HEK cells transfected with a reporter plasmid containing a chimeric firefly luciferase with the Muc5b 3′ UTR and either a control or miR-34b-5p. *P < 0.05; Fluc/Rluc (firefly luciferase/renilla luciferase). (E) Predicted miR-144-3p binding site in the Tgfbr1 3′ UTR region was identified with http://www.microrna.org/microrna/home.do (F) Tgfbr1 immunoblot of MLE-12 cells transfected with the control or miR-144-3p mimics. (G) Luciferase activity of HEK cells transfected with a reporter plasmid containing a chimeric firefly luciferase with the Tgfbr1 3′ UTR and either a control or miR-144-3p. *P < 0.05. (H) TGF-β signaling regulatory network of experimentally identified targets of miR-34b-5p (blue), miR-503-5p (purple), miR-144-3p (brick red), and miR-142-3p (orange). These curated targets were experimentally validated either by UTR analysis (closed circle) or with pull-down methods (dotted circle). (I) Immunoblot for phosphorylated SMAD2 and SMAD2 using MLE-12 cells transfected with control or miR-144-3p mimics followed by TGF-β stimulation. (J) Wnt signaling regulatory network of experimentally identified targets (labeling identical to H). (K) Luciferase activity of HEK cells cotransfected with a TopFlash reporter plasmid and miRNAs as labeled. *P < 0.05; **P < 0.005; ***P < 0.0005 by 1-way ANOVA analysis.