Sp1 Mediates a Therapeutic Role of MiR-7a/b in Angiotensin II-Induced Cardiac Fibrosis via Mechanism Involving the TGF-β and MAPKs Pathways in Cardiac Fibroblasts

<div><p>MicroRNA-7a/b (miR-7a/b) protects cardiac myocytes from apoptosis during ischemia/reperfusion injury; however, its role in angiotensin II (ANG II)-stimulated cardiac fibroblasts (CFs) remains unknown. Therefore, the present study investigated the anti-fibrotic mechanism of miR-7a/b in ANG II-treated CFs. ANG II stimulated the expression of specific protein 1 (Sp1) and collagen I in a dose- and time-dependent manner, and the overexpression of miR-7a/b significantly down-regulated the expression of Sp1 and collagen I stimulated by ANG II (100 nM) for 24 h. miR-7a/b overexpression effectively inhibited MMP-2 expression/activity and MMP-9 expression, as well as CF proliferation and migration. In addition, miR-7a/b also repressed the activation of TGF-β, ERK, JNK and p38 by ANG II. The inhibition of Sp1 binding activity by mithramycin prevented collagen I overproduction; however, miR-7a/b down-regulation reversed this effect. Further studies revealed that Sp1 also mediated miR-7a/b-regulated MMP expression and CF migration, as well as TGF-β and ERK activation. In conclusion, miR-7a/b has an anti-fibrotic role in ANG II-treated CFs that is mediated by Sp1 mechanism involving the TGF-β and MAPKs pathways.</p></div

Effect of different concentrations of mithramycin on the regulation of Sp1, collagen Iexpression and signal transduction in CFs.

<p>A-F: Western blots and analysis of mithramycin on the regulation of Sp1 (A), collagen I (B), TGF-β (C), p-ERK (D), p-JNK (E) and p-p38 (F). Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; and #p < 0.05, compared with NC.</p

Effect of miR-7a/b mimics on TGF-β and MAPK pathways.

<p>Western blots and analysis of TGF-β (A), p-ERK (B), p-JNK (C), and p-p38 (D) expression. Con: normal untreated CFs; NC: negative control siRNA; *p < 0.05, compared with control; and #p, < 0.05 compared with NC.</p

Effect of miR-7a/b mimics on proliferation, migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A: MTT assay. B-C: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. D-F: Western blot analysis of MMP-2 (D, E) and MMP-9 (D, F) protein levels. G-H: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in the media. Con: normal untreated CFs; NC: negative control siRNA; *p, < 0.05 compared with control; and #p, < 0.05 compared with NC.</p

Sp1 mediates miR-7a/b-regulated proliferation migration, and MMP-2 and MMP-9 expression/activity in CFs.

<p>A-C: Western blot analysis of MMP-2 (A, B) and MMP-9 (A, C) protein levels. D-E: Media were harvested for gelatin zymography analysis of MMP-2 activity; no MMP-9 activity was detected in media. F: MTT assay. G-H: CFs on the external surface of the Transwell were dyed with Crystal Violet and photographed under a microscope. Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; #p < 0.05, compared with NC; and ◆p < 0.05, compared with mithramycin.</p

Effect of miR-7a/b mimics on the regulation of Sp1 and collagen Iexpression and localization in CFs.

<p>A: Western blots of Sp1 and collagen I in untreated CFs and ANG II-treated CFs that were treated with NC siRNA or miR-7a/b mimics. B: Immunofluorescent staining of Sp1 and collagen I location in normal untreated CFs and ANG II-treated CFs that were treated with NC siRNA or miR-7a/b mimics. Con: normal untreated CFs; NC: negative control siRNA. *p < 0.05, compared with control; and #p < 0.05, compared with NC.</p

Sp1 mediates miR-7a/b-regulated collagen I expression and signal transduction in CFs.

<p>A-D: Western blots and analysis of Sp1-mediated miR-7a/b-regulated expression of Sp1 (A), collagen I (B), TGF-β (C) and p-ERK (D). Con: normal untreated CFs; NC: negative control siRNA; M: mithramycin. *p < 0.05, compared with control; and #p, < 0.05 compared with NC; and ◆p < 0.05, compared with mithramycin.</p

ANG II stimulated Sp1 and collagen I expression in CFs.

<p>A: Protein expression of Sp1 and collagen I in 100 nM ANG II-treated CFs at different time points. B: Protein expression of Sp1 and collagen I treated with different concentrations of ANG II for 24 h. C: normal untreated CFs. *p < 0.05, compared with control.</p