MicroRNA-1-Mediated Inhibition of Cardiac Fibroblast Proliferation Through Targeting Cyclin D2 and CDK6

Figures

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  Figure 1. MiRNA-1 is down-regulated upon cardiac fibroblast activation in vivo from subcutaneous Ang II-infused hearts and in vitro upon Ang II or TGFβ stimulation. (A) Representative images of Picrosirius Red staining from free wall (cross sections) of left ventricles of control (saline) and Ang II-infused hearts. Scale bar: 500 μm. (B) Quantitation of Picrosirius Red staining in entire ventricular cross-section area. n = 5 rats each. Data are expressed as relative changes to Ctr. *P < 0.05 vs. Ctr. (C) Heart weight (HW) to body weight (BW) ratios. n = 5–6 rats each. * P < 0.05 vs. Ctr. (D) Real-time PCR analysis of ANF mRNA expression in freshly isolated cardiac myocytes from rats subjected to subcutaneous saline (Ctr) or Ang II infusion. Data are normalized to 18S and expressed relative to controls. n = 8–9 rats each, *P < 0.05 vs. Ctr. (E,F) Real-time PCR analysis of miRNA-1 expression in freshly isolated cardiac myocytes and cardiac fibroblasts from rats subjected to subcutaneous saline (Ctr) or Ang II infusion for 4 weeks. Data are normalized to U6 and expressed relative to controls. n = 7 rats each. * P < 0.05 vs. Ctr. (G) Real-time PCR analysis of miRNA-1 expression in adult rat…

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  Figure 2. MiRNA-1 negative regulates cardiac fibroblast proliferation. (A) MiRNA-1 level in adult rat ventricular fibroblasts infected with Ad-Ctr or Ad-miRNA-1 (MOI 20) for 48 h. Data are normalized to U6 and expressed relative to controls. n = 6 each, * P < 0.05 vs. Ad-Ctr. (B) MiRNA-1 level in adult rat ventricular fibroblasts transfected with negative control (Neg Ctr) or miRNA-1 antagomirs for 48 h. Data are normalized to U6 and expressed relative to controls. n = 6 each. *P < 0.05 vs. Neg Ctr antagomir. (C) Basal and TGFβ1 (10 ng/ml, 48 h)-induced cardiac fibroblast proliferation 72 h after Ad-Ctr or Ad-miRNA-1 infections (MOI 20). Data are expressed as relative changes to controls under basal conditions. n = 7 each. *P < 0.05 vs. Ad-Ctr. (D) Basal and TGFβ1 (10 ng/ml, 48 h)-induced cardiac fibroblast proliferation 72 h after transfection with negative control (Neg Ctr) or miRNA-1 antagomirs. Data are expressed as relative changes to controls under basal conditions. n = 6 each. *P < 0.05 vs. Neg Ctr antagomir.

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  Figure 3. MiRNA-1 directly targets cyclin D2 and CDK6. (A,B) The sequences of putative miRNA-1 target sites as predicted by TargetScan and the conserved seed sequences are shown. (C,D) Normalized luciferase ratios in fibroblasts that were infected with Ad-Ctr or Ad-miR-1 (MOI 20) overnight, followed by transfection with plasmid constructs containing wild type (WT), mutation (MUT), or deletion (DEL) of the 3′-UTR binding site of the indicated putative target for 48–72 h. n = 6 each. * P < 0.05 vs. Ad-Ctr. (E,F) Western blots showing cyclin D2 and CDK6 expression in fibroblasts infected with Ad-Ctr or Ad-miRNA-1 (MOI 20) for 29 h. Representative images in each row are from the same membrane; spaces indicate removal of lanes with other conditions. Graphs showing quantitative densitometry and relative to Ad-Ctr. n = 6 each. * P < 0.05 vs. Ad-Ctr. (G,H) Real-time PCR analysis of cyclin D2 and CDK6 mRNA levels in fibroblasts infected with Ad-Ctr or Ad-miRNA-1 (MOI 20) for 48 h. Data are normalized to 18S and expressed relative to controls. n = 6 each. * P < 0.05 vs. Ad-Ctr.

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  Figure 4. MiRNA-1 regulates cell cycle regulators and their downstream signaling in cardiac fibroblasts and overexpression of cyclin D2 blunts miRNA-1-mediated inhibition of cardiac fibroblast proliferation. (A) Scheme showing the working model of miRNA-1-mediated inhibition of cardiac fibroblast proliferation through targeting cyclin D2 and CDK6. (B,C) Western blots probed with the indicated antibodies. Cardiac fibroblasts were treated with or without TGFβ1 (10 ng/ml) for 48 h after Ad-Ctr or Ad-miRNA-1 infection (MOI 20) for 24 h. Graphs showing quantitative densitometry with relative changes to Ad-Ctr at basal condition. n = 6 each. * P < 0.05 for the indicated comparisons. (D,E) Western blots showing phosphorylation levels of Rb protein at Ser780 and Ser795 in cardiac fibroblasts that were treated with or without TGFβ1 (10 ng/ml) for 29 h after Ad-Ctr or Ad-miRNA-1 infection (MOI 20) for 24 h. Graphs showing quantitative densitometry with relative changes to Ad-Ctr at basal condition. n = 6 each. * P < 0.05 for the indicated comparisons. (F) Western blot showing overexpression level of HA-tagged cyclin D2 in cardiac fibroblasts after infection with Ad-Ctr or Ad-cyclin D2 (MOI 10) for…

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  Figure 5. MiRNA-1's targets (cyclin D2 and CDK6) are up-regulated together with increase of cell proliferation marker PCNA in cardiac fibroblasts activated in vivo via subcutaneous Ang II-infusion for 4 weeks. (A,B) Western blots using cell lysates from freshly isolated cardiac fibroblasts from rats subjected to subcutaneous saline (Ctr, n = 3–5 rats) or Ang II infusion (n = 4 rats) for 4 weeks. Graphs showing quantitative densitometry of Western blots expressed as relative changes to Ctr. * P < 0.05 vs. Ctr.