miR-96-5p regulates myocardial infarction-induced cardiac fibrosis via Smad7/Smad3 pathway

Figure 1 Increased miR-96-5p is associated with cardiac fibrosis (A) miR-seq analysis of cardiac samples from mice undergoing sham and MI surgeries ( n=3). (B) The relative mRNA level of α-SMA ( n=5). (C) qPCR analysis of miR-96-5p expression ( n=5). (D,E) The relative α-SMA mRNA and miR-96-5p expression levels of two in vitro cardiac fibrosis models (NRCFs stimulated with 20 ng/mL TGF-β or 200 nM Ang II for 48 h, n=5). (F) miR-96-5p levels in NRCFs versus NRCMs ( n=5). (G) Expression of miR-96-5p in NRCMs transfected with miR-96-5p mimic or inhibitor ( n=5). (H) The relative mRNA levels of BNP, Col1a1, and Col3a1 in NRCMs transfected with miR-96-5p mimic or inhibitor ( n=5). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 2 Inhibition of miR-96-5p alleviates myocardial fibrosis in post-MI mice (A) qPCR analysis of miR-96-5p expression in the heart chamber ( n=5). (B) Mouse cardiac function was measured by EF%, FS%, LVIDs, and LVIDd ( n=5). (C) Images and measurement of Masson’s trichrome-stained cardiac sections ( n=5). Upper scale bar: 1 mm; lower scale bar: 200 μm. (D) The relative mRNA levels of Col1a1, Col3a1, and α-SMA in ventricular tissues ( n=5). (E) Protein levels of Col1a1, Col3a1, CTGF, and α-SMA in ventricular tissues determined by western blot analysis ( n=5). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 3 Effect of miR-96-5p mimic on the fibrosis degree of NRCFs stimulated by TGF-β (A) Expression of miR-96-5p in NRCFs transfected with mimic ( n=5). (B) Relative mRNA levels of fibrosis-associated genes normalized to 18 s in NRCFs ( n=5). (C) NRCF proliferation was evaluated using EdU, and NRCF differentiation was evaluated using immunofluorescence staining of α-SMA ( n=5). Scale bar: 200 μm. (D) Western blot analysis was performed to determine the protein levels of Col1a1 and α-SMA in NRCFs ( n=6). ** P<0.01, *** P <0.001 vs corresponding controls.

Figure 4 Effect of miR-96-5p mimic on NRCFs in the presence of Ang II (A) qPCR was used to detect the mRNA levels of profibrotic genes in NRCFs ( n=5). (B) Proliferation and differentiation of NRCFs determined by immunofluorescence staining of EdU and α-SMA ( n=5). Scale bar: 200 μm. (C) Protein expressions of Col1a1 and α-SMA in NRCFs ( n=6). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 5 miR-96-5p inhibition suppresses the level of fibrosis in NRCFs in the presence of TGF-β (A) Expression of miR-96-5p in NRCFs transfected with inhibitor ( n=5). (B) Relative mRNA levels of Col1a1, Col3a1, and α-SMA normalized to 18 s in NRCFs ( n=5). (C) Proliferation and differentiation degree of NRCFs evaluated by EdU/α-SMA staining ( n=5). Scale bar: 200 μm. (D) Western blot analysis of the protein levels of fibrosis-related genes in NRCFs ( n=6). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 6 miR-96-5p inhibitor impedes the proliferation and differentiation abilities of NRCFs treated with Ang II (A) Relative mRNA levels of Col1a1, Col3a1, and α-SMA detected by qPCR ( n=5). (B) NRCF proliferation was indicated by EdU staining, while NRCF differentiation was indicated by α-SMA staining ( n=5). Scale bar: 200 μm. (C) Protein levels of fibrosis-associated genes in NRCFs detected by western blot analysis ( n=6). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 7 Smad7 is a direct target of miR-96-5p (A) Potential targets of miR-96-5p were predicted using three databases. (B) Smad7 was identified as the direct target of miR-96-5p by dual luciferase reporter assay ( n=3). (C) Protein level of Smad7 in NRCFs treated with miR-96-5p mimic or inhibitor ( n=6). (D) Protein level of Smad7 in NRCFs stimulated with TGF-β or Ang II ( n=6). (E) The silencing efficiency of Smad7 siRNA was tested by qPCR and western blot analysis ( n=5). ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 8 Smad7 knockdown attenuates the anti-fibrotic effect of the miR-96-5p inhibitor on NRCF (A) mRNA levels of fibrosis-related genes in NRCFs detected by qPCR ( n=5). (B) The proliferation and differentiation abilities of NRCFs were assessed by EdU/α-SMA staining ( n=5). Scale bar: 200 μm. (C) Protein levels of Col1a1 and α-SMA in NRCFs ( n=6). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.

Figure 9 Smad7 mediates the effect of miR-96-5p by enhancing Smad3 phosphorylation (A) Knockdown of Smad7 activated Smad3 phosphorylation ( n=6). (B) Protein levels of p-Smad3 and Smad7 in ventricular tissues of sham and MI mice ( n=5). (C−E) The Smad3 inhibitor weakened the effect of the miR-96-5p mimic on NRCFs, as evidenced by qPCR ( n=5), EdU/α-SMA staining ( n=5), and western blot analysis ( n=6). * P<0.05, ** P<0.01, *** P<0.001 vs corresponding controls.