Abstract 318: Targeting of Tp53inp1 by Mir-221 to Reduce P62-mediated Autophagy is Cardioprotective in Ischemia / Reperfusion Injury
Purpose: Tumor protein 53-induced nuclear protein 1 (Tp53inp1) acts as a tumor suppressor by inducing cell death. Tp53inp1 mRNA is a predicted target of miR-221. Whether targeting Tp53inp1 plays a role in miR-221-mediated cardioprotection has not been investigated. We hypothesized that miRNA-221 directly targets Tp53inp1 to reduce ischemia/reperfusion (I/R)-induced autophagy.
Method: Myoblast H9c2 cells underwent 16 hours 0.2% O2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R). H9c2 were transfected with miRNA-221 mimic (25 nmol) and scrambled mimic control (miR-221 and MC). Cell count/viability, WST assay, cell injury-induced LDH release, and GFP-LC3 labeled autophagosome formation were measured. Cells were collected for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Tp53inp1 and pcDNA3.1-Flag-p62 plasmids were cloned and transfected into H9c2 for recovery and immuno-precipitation (IP) studies. The effects of miRNA-221 inhibitor in H9c2 were also assessed.
Results: miR-221 significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-221 vs. MC p<0.05). qPCR confirmed that (1) miRNA-221 expression was reduced in H-R; (2) RISC-loaded (IP pull-down Ago-2) miRNA-221 increased by ~80 fold and reduced by 95% following mimic and inhibitor transfection respectively; (3) Increased Tp53inp1 following H-R was reversed by miR-221. miR-221 inhibited H-R induced autophagosome formation (GFP-LC3). WB indicated (1) increase of LC3-I/II ratio and p62, indicators of reduced autophagy, and (2) decrease of Tp53inp1 by miR-221. IP pull-down Myc-Tp53inp1 indicated the formation of p62-Tp53inp1 complex. The protective effect of miR-221 was abolished by Tp53inp1 overexpression (pCMV-Myc-Tp53inp1 and miRNA-221 mimic co-transfection). The protective effect was corroborated in neonatal rat ventricular myocytes (NRVM). MiRNA-221 inhibitor induced reverse effects.
Conclusion: The cardioprotection of miR-221 entails direct targeting of Tp53inp1 which reducing p62-Tp53inp1 complex formation and inhibiting H-R-induced autophagy.
Author Disclosures: P. Wang: None. Q. Chen: None. A.M. Richards: None.
- © 2015 by American Heart Association, Inc.