Abstract 18392: Mir-101a and Tgf-β Are Mutually Inhibitory: Escape of Tgf-β From Mir-101a Inhibition Promotes Cardiac Fibrosis
MiR-101a is a potent cancer suppressor. Its down-regulation promotes cancer cell proliferation and migration. Conversely miR-101a upregulation inhibits liver and lung fibrosis. Its role in cardiac fibrosis is unknown. We hypothesized miR-101a functions as an endogenous TGF-β signaling inhibitor and that escape from such inhibition leads to cardiac fibrosis. Cardiac fibroblasts (FB) were isolated from adult rat hearts, transfected with miR-101a mimic or inhibitor and then treated with TGF-β, Balfilomycin (Baf) or Rapamycin (Rap). Cell proliferation assay CCK8 and myofibroblast marker α-SMA were measured. LC3, p62 and mTOR signaling were assessed by Western blot. Apoptosis was assessed by MUSE cell analyzer with 7-AAD and Annexin V staining. Left coronary artery ligation (MI) was performed. Hearts were collected and underwent picro-sirius staining and stem-loop qPCR.
Result: TGF-β caused downregulation of miR-101a in FB and concurrent fibrosis as indicated by increased CCK8, α-SMA expression and gel contraction. Interestingly, autophagy was reduced as indicated by falls in LC3 II/I ratio and apoptosis was also reduced. Transfection with miR-101a mimic had opposite effects to TGF-β and miR-101a inhibitor had similar effects. Autophagy flux test, with additional Baf treatment, indicated that TGF-β reduced, while miR-101a augmented, autophagosome formation and degradation. MiR-101 overexpression was associated reduced phosphorylation of p70s6p and 4EBP1, suggesting modulation of mTOR signaling and its downstream effectors. Modulation of autophagy by Rap or Baf respectively accelerated or reversed miR-101a-induced inhibition of FB proliferation. TGF-β-induced FB proliferation was completely blocked by miR-101a. MiR-101a was reduced in the infarct and border zone but unaltered in the remote zone 7 days after MI. This change was correlated with fibrosis (scare formation in the infarct zone) in different zones.
Conclusion: miR-101a is an endogenous TGF- β inhibitor. Escape of TGF- β from the inhibitory effects of miR-101a lead to cardiac fibrosis. Underlying mechanisms may relate to direction of FB fate through regulation of autophagy and apoptosis. MiR-101a is a promising therapeutic target for treatment of cardiac fibrosis.
Author Disclosures: Y. ZHou: None. A.M. Richards: None. P. Wang: None.
- © 2016 by American Heart Association, Inc.