Hypoxic myocardial injury model
The rat myocardial cell line H9C2 (American Type Culture Collection, Manassas, USA) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco-BRL, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, USA). All cells were maintained in a humidified incubator containing 95% air and 5% CO2 at 37°C. Cells were cultured to 70-80% confluence, which was determined using a phase contrast microscopy (Olympus, Tokyo, Japan). After reaching 70%–80% confluence, H9C2 cells were treated with 400 μM CoCl2for 24, 48 and 72 h.
Cell transfection Hypoxic H9C2 cells were seeded at a density of 3 × 104 cells/well in six-well plates and allowed to adhere to the dish overnight. The next day, the miR-negative control (NC), miR-210 mimic and miR-210 inhibitor (GenePharma Co, Shanghai, China) were transfected into H9C2 cells using Lipofectamine 2000 reagent (Life Technologies, Gaithersburg, USA) according to manufacturer’s protocol.