Abstract 319: Mirna-125b Reduces Ischemia / Reperfusion (i/r)-induced Apoptosis Through Targeting Multiple Targets in Intrinsic and Extrinsic Apoptosis Pathways
Apoptosis is mediated through extrinsic and intrinsic pathways, both play a role in ischemia/reperfusion (I/R) injury. Predicted targets for miRNA-125b include extrinsic pathway mediators Traf6 and Tnfrsf1b, and intrinsic mitochondria regulators Bcl-2 family pro-apoptotic effectors Bak1 and BH3-only facilitators Bim, Bmf, Puma. We hypothesized that miRNA-125b directly targets multiple genes to reduce I/R-induced apoptosis. Myoblast H9c2 cells underwent 16 hours 0.2% O2 hypoxia followed by 2 hours re-oxygenation (H-R, simulating I/R) and were transfected with miRNA-125b mimic vs. scrambled mimic control (25 nmol, miR-125b-M vs. MC) and miR-125b inhibitor vs. inhibitor control (miR-125b-I vs. IC). Cell count/viability, WST assay, cell injury-induced LDH release and apoptotic marker Casp3/7 were measured. Cells were trypsinized for assessment of apoptosis (7-AAD and annexin V double staining) and lysed for RT-qPCR and western blot (WB) analyses. pCMV-Myc-Bak1 plasmids were cloned and transfected into H9c2 for recovery studies. The effects were verified in neonatal rat ventricular myocytes (NRVM). miRNA-125b-M significantly reduced H-R injury as indicated by higher cell count/viability and WST activity, and reduced LDH (miR-125b-M vs. MC p<0.05). qPCR confirmed that (1) miR-125b expression was reduced in H-R; (2) RISC-loaded (immunoprecipitation pull-down Ago-2) miR-125b increased by ~35 fold and reduced to ~3% following mimic and inhibitor transfection respectively; (3) multiple apoptosis-related genes were reduced by miR-125b-M, Bak1, Bmf, Bim, Puma, Traf6 and Tnfrsf1b. All changes were confirmed by WB. Luciferase reporter assays indicated miR-125b bound to the 3’-UTR of all genes tested except Traf6. Total apoptotic cell numbers and Casp3/7 release were significantly reduced by miR-125b-M. The protective effect of miRNA-125b was partially abolished by Bak1 overexpression (pCMV-Myc-Bak1 and miR-125b co-transfection). Protective effects of miRNA-125b were further verified in NRVM. MiRNA-125b inhibitor reversed protective effects and target changes at mRNA and protein level. miR-125b is powerfully cardioprotective in I/R injury due to directly targeting multiple genes in the extrinsic and intrinsic apoptotic pathways.
Author Disclosures: P. Wang: None. Q. Chen: None. Y. Zhou: None. A.M. Richards: None.
- © 2015 by American Heart Association, Inc.