Neonatal rat cardiac fibroblasts (NRCFs) were isolated from newborn SD rats (1-3 day). First, minced ventricle tissues were digested in the buffer of 40% collagenase and 60% trypsin (BioFroxx, Guangzhou, China). Then, NRCFs were separated from cardiomyocytes by gravity separation for 2 h, attached to the 10 cm cell culture dishes, and cultured with medium (DMEM+10% fetal bovine serum+1% streptomycin+1% penicillin) (Gibco, Grand Island, CA, USA). Cells were kept at 37°C in humid air containing 5% CO2.
Cells at the second passage were exposed to miR-409-3p mimic (100 nM)/inhibitor (200 nM) versus the corresponding nc mimic/nc inhibitor (RiboBio) for 48 hours in culture medium with 1% fetal bovine serum (FBS). At the same time, cells were treated with Ang II (200 nM, 48 h, Sigma, St. Louis, MO, USA) or recombinant human TGF-β1 (20 ng/mL, 48 h, PeproTech, Rocky Hill, NJ, USA). Reagents above including Gpd1 siRNA, GATA2 siRNA, and nc siRNA (50 nM, 48 h, Sangon, Shanghai, China) were all transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
Cells at the second passage were exposed to miR-409-3p mimic (100 nM)/inhibitor (200 nM) versus the corresponding nc mimic/nc inhibitor (RiboBio) for 48 hours in culture medium with 1% fetal bovine serum (FBS). At the same time, cells were treated with Ang II (200 nM, 48 h, Sigma, St. Louis, MO, USA) or recombinant human TGF-β1 (20 ng/mL, 48 h, PeproTech, Rocky Hill, NJ, USA). Reagents above including Gpd1 siRNA, GATA2 siRNA, and nc siRNA (50 nM, 48 h, Sangon, Shanghai, China) were all transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.