HIF-1a-induced upregulated miR-322 forms a feedback loop by targeting Smurf2 and Smad7 to activate Smad3/beta-catenin/HIF-1 alpha, thereby improving myocardial ischemia-reperfusion injury

Myocardial ischemia/reperfusion injury (MIRI) is a major cause of heart failure after myocardial infarction. It has been reported that miR-322 is involved in MIRI progression, while the molecular mechanism of miR-322 in regulating MIRI progression needs to be further probed. MIRI cell model was established by oxygen-glucose deprivation/reoxygenation (OGD/R). Cell viability was assessed using MTS assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining were employed to analyze cell apoptosis. In addition, the interactions between miR-322, Smad7/Smurf2, hypoxia-inducible factor alpha (HIF-1 alpha), and beta-catenin were verified by dual-luciferase reporter gene assay. Our results displayed that miR-322 was significantly downregulated in OGD/R-treated H9c2 cells, and its overexpression resulted in increased cell viability and reduced the apoptosis. Smurf2 and Smad7 were identified as the direct targets of miR-322. Smad7 knockdown or Smurf2 knockdown increased OGD/R-treated H9c2 cell viability and suppressed the apoptosis. Meanwhile, miR-322 mimics abolished the mitigating effect of Smad7 or Smurf2 overexpression on MIRI. In addition, the Smad3/beta-catenin pathway was identified as the downstream pathway of Smurf2/Smad7. Moreover, it was found that HIF-1 alpha interacted with the miR-322 promoter, and beta-catenin interacted with the HIF-1a promoter to form a loop. HIF-1 alpha-induced upregulated miR-322 activated the Smad3/beta-catenin pathway by targeting Smurf2 and Smad7 to improve MIRI; meanwhile, beta-catenin/HIF-1 alpha formed a positive feedback loop to continuously improve MIRI.