Fig 1: Smad7 is identified as an essential target of miR-25 in cardiac fibrosis(A) The alignment results between miR-25 and Smad7 3′-UTR provide insights into the specific binding locations within the genome sequence. (B) Luciferase reporter activity was measured using Smad7 3′UTR in a dose-dependent manner with miR-25 mimic. Additionally, a deletion mutation in the miR-25 binding site of Smad7 3′UTR was applied to validate the luciferase activity. Protein levels of SERCA2a and Smad7 were examined by western blot analysis after AAV9 pre-miR-25 delivery in WT mice (C), or lentiviral shSmad7 vector delivery (D). (E) AAV9 miR-25 TuD was injected into mdx/utrn (+/−) mice, with or without the lentiviral shSmad7 vector, and protein levels of SERCA2a and Smad7 were detected by western blot analysis. (F) Following euthanasia, mouse hearts were cryosectioned and subjected to Masson’s trichrome staining for the evaluation of cardiac fibrosis. In the western blot analysis, 15 μg of proteins was loaded into each well. For (A)–(F), the data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005 (n = 3 for each group).
Fig 2: Direct intramuscular injection of AAV9 miR-25 TuD improves muscle performance(A) The treadmill exhaustion test involved calculating the average running distance on the treadmill during the exercise program. The bars represent the mean values for each group, with error bars indicating the standard deviation (n = 8 for each group). (B) Representative histological images with H&E and picrosirius red staining depict mononuclear infiltrates and tissue fibrosis. The scale bar indicates 100 μm. (C) Ultrastructural images were obtained using transmission electron microscopy. (D) Representative western blot images evaluating markers such as SERCA2a for cardiac calcium pump, vimentin, fibronectin, and gelsolin for fibrosis, TGF-β1, p-Smad2, total Smad2/3, and Smad7 for TGF-β signaling molecules, and α-SMA for myofibroblast (n = 3 for each group). (E) Pre- and mature forms of miR-25 were measured before and after gene transfer (n = 5 for each group). The mRNA expressions for (F) anti-inflammatory and (G) pro-inflammatory cytokines were measured using the qRT-PCR method in skeletal muscles (n = 5 for each group). In the western blot analysis, 20 μg of proteins was loaded into each well. For (A)–(G), the data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.
Fig 3: AAV9 miR-25 TuD reduces fibrosis by inhibiting TGF-β signaling pathways by restoring Smad7 expressionWestern blotting was performed to evaluate the expression of Smad7 and GAPDH in the canine (GRMD) models (A), with n = 2 for each group. Similarly, in the mdx/utrn (+/−) mouse models, western blot analysis was conducted to assess Smad7 and GAPDH expression (B), with n = 5 for each group. (C) The expression levels of pre- and mature miR-25 were quantified using qRT-PCR in mdx/utrn (+/−) mice treated with AAV9 control or AAV9 miR-25 TuD, with n = 5 for each group. (D) Furthermore, qRT-PCR analysis was employed to analyze the mRNA expression levels of fibrosis markers, such as fibronectin, galectin-3, and TGF-β1, in comparison with the WT group. In the western blot analysis, 20 μg of proteins was loaded into each well. For (A)–(D), the data represent the mean ± SD. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.
Supplier Page from OriGene Technologies for Smad7 Mouse shRNA Lentiviral Particle (Locus ID 17131)