miR-7a/b attenuates post-myocardial infarction remodeling and protects H9c2 cardiomyoblast against hypoxia-induced apoptosis involving Sp1 and PARP-1

Abstract

miRs (microRNAs, miRNAs) intricately regulate physiological and pathological processes. Although miR-7a/b protects against cardiomyocyte injury in ischemia/reperfusion injury, the function of miR-7a/b in myocardial infarction (MI)-induced cardiac remodeling remains unclear. Here, we sought to investigate the function of miR-7a/b in post-MI remodeling in a mouse model and to determine the underlying mechanisms involved. miR-7a/b overexpression improved cardiac function, attenuated cardiac remodeling and reduced fibrosis and apoptosis, whereas miR-7a/b silencing caused the opposite effects. Furthermore, miR-7a/b overexpression suppressed specific protein 1 (Sp1) and poly (ADP-ribose) polymerase (PARP-1) expression both in vivo and in vitro, and a luciferase reporter activity assay showed that miR-7a/b could directly bind to Sp1. Mithramycin, an inhibitor of the DNA binding activity of Sp1, effectively repressed PARP-1 and caspase-3, whereas knocking down miR-7a/b partially counteracted these beneficial effects. Additionally, an immunoprecipitation assay indicated that hypoxia triggered activation of the binding activity of Sp1 to the promoters of PARP-1 and caspase-3, which is abrogated by miR-7a/b. In summary, these findings identified miR-7a/b as protectors of cardiac remodeling and hypoxia-induced injury in H9c2 cardiomyoblasts involving Sp1 and PARP-1.

Figure 1. Expression levels of miR-7a/b fluctuated post MI and miR-7a/b overexpression improved cardiac function.

(A,B) RT-PCR results represents the expression levels of miR-7a/b in the border zone of the hearts at different time after MI. (C) Representative 2D echocardiograms and M-mode echocardiograms 4 weeks after MI. (D) Left ventricular ejection fraction (LVEF). (E) Fractional shortening (FS). (F) Diastolic left ventricular internal diameter LVIDd. (G) Systolic left ventricular internal diameter (LVIDs). Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. D: Days after MI. Data are the mean ± SD, n = 3–5/group (A,B), n = 6–7/group (C–G), *p < 0.05 compared with Con, ^#p < 0.05 compared with GFP-NC.

Figure 2. miR-7a/b overexpression ameliorated myocardial fibrosis.

(A) Representative Masson’s trichrome staining (first row), Picrosirius red staining (second and third row), immunostaining of collagen I (fourth row) and collagen III (fifth row) (scale bar: 20 μm). (B) Quantitative analysis of myocardial fibrosis. (C–F): Western blots analysis of protein expression of collagen I and collagen III. Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. (D) Days after MI. Data are the mean ± SD, n = 6–7/group (A,B), n = 3–5/group (C–F), *p < 0.05 compared with Con, ^#p < 0.05 compared with GFP-NC.

Figure 3. miR-7a/b overexpression reduced apoptosis of the heart.

(A) Representative apoptosis cells in the border zones of the hearts post MI (scale bar: 20 μm). (B) Analysis of apoptosis cells. Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. (D) Days after MI. Data are the mean ± SD, n = 6–7/group, *p < 0.05 compared with Con, ^#p < 0.05 compared with GFP-NC.

Figure 4. miR-7a/b repressed of PARP-1 expression in vivo and in vitro, and reduced apoptosis in vitro.

(A) Immunostaining of PARP-1 in vivo. (B) Western blots of PARP-1 in vivo. (C) Western blots of PARP-1 in cells exposed to hypoxia for different time. (D–G) Western blots showing the effect of miR-7a/b on regulation of PARP-1 (D,E), cleaved caspase-3 (D,F), Bax/Bcl-2 (D,G). (H,I) TUNEL assay results (Scale bar: 50 μm). Con: sham mice without LAD occlusion (A,B) or normal cultured H9c2 cells (C–H). MI: mice with LAD occlusion. NC: H9c2 cells exposed to hypoxia for 12 h. Data are the mean ± SD, n = 6/group (A), n = 3/group (B–I), *p < 0.05 compared with Con; ^#p < 0.05 compared with GFP-NC (A,B) or NC (C–I).

Figure 5. 3-AB decresed PARP-1 expression and apoptosis in vitro.

(A–D) Western blots showing the effect of 3-AB on regulation of PARP-1 (A,B), cleaved caspase-3 (A,C), Bax/Bcl-2 (A,D). (E,F) TUNEL assay results (Scale bar: 50 μm). Con: normal cultured H9c2 cells. –, cells only exposed to hypoxia. Data are the mean ± SD, n = 3/group, *p < 0.05 compared with Con; ^#p < 0.05 compared with-.

Figure 6. miR-7a/b repressed the downstream target Sp1 expression in vivo and in vitro.

(A) Western blots of Sp1 in cells exposed to hypoxia for different time, (B,C) Western blots showing the effect of miR-7a/b on regulation of Sp1 in vitro (B) and in vivo (C). (D) Immunostaining of Sp1 in vivo (scale bar: 20 μm). (E) Conserved miR-7a/b binding sites and mutated binding sites in 3′ untranslated region (UTR) of Sp1. (F) Luciferase activity analysis. Con: sham mice without LAD occlusion. MI: mice with LAD occlusion. Con: normal cultured H9c2 cells (A,B) or sham mice without LAD occlusion (C,D). MI: mice with LAD occlusion. NC: H9c2 cells exposed to hypoxia for 12 h. Data are the mean ± SD, n = 3/group (A–C,F), n = 6/group (D), *p < 0.05 compared with Con (A–C) or NC siRNA. (F); ^#p < 0.05 compared with NC (A,B) or GFP-NC (C,D).

Figure 7. Sp1 binding activity mediated miR-7a/b-regulated Sp1, PARP-1 and caspase-3 expression in hypoxia H9c2 cells.

(A,D–F) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic cells pretreated with different concentration of mithramycin (nM). (C,G–I) Western blots of Sp1, PARP-1 and caspase-3 in hypoxic H9c2 cells transfected with miR-7a/b inhibitors that treated with or without 100 nM mithramycin. (B) Representative ChIP assays. Con: normal cultured H9c2 cells, M: mithramycin, *p < 0.05 compared with control group, ^#p < 0.05 compared with NC siRNA-transfected group, ^♦p < 0.05 compared with miR-7a/b inhibitors-transfected group; ^Φp < 0.05 compared with mithramycin-treated group.