Mir 582 5p inhibitor

The cell transfection procedures were performed as previously described.⁴¹ MiR‐582‐5p inhibitor, miR‐582‐5p mimic, small interfering RNA (siRNA) against Creb1 (siRNA), and the corresponding negative controls were purchased from RIBOBIO. Lipofectamine 3000 transfection reagent was used for all transfections procedures according to the manufacturerʼs instructions (Cat: L3000015; Invitrogen).⁴² Briefly, HL‐1 cells were seeded into six‐well plates overnight until they reached 50%–60% confluence. Oligonucleotides were mixed into lipofectamine 3000 reagents for 5 min at RT, followed by the addition of the Opti‐MEM to a final concentration of 50 nM. The cells were subjected to OGD after transfection for 24 h. The corresponding oligonucleotides sequences are as follows:
Creb1‐siRNA‐F: 5′‐GGCUAACAAUGGUACGGAUTT‐3′, Creb1‐ siRNA‐R: 5′‐AUCCGUACCAUUGUUAGCCTT‐3′; miR‐582‐5p mimic‐F: 5′‐AUACAGUUGUUCAACCAGUUAC‐3′, miR‐582‐5p mimic‐R: 5′‐AACUGGUUGAACAACUGUAUUU‐3′, MiR‐582‐5p inhibitor: 5′‐GUAACUGGUUGAACAACUGUAU‐3′.

Before transfection, shF2RL2, shRNA for lncRNA NEAT1 (shlncNEAT1), shNC, miR-582-5p mimic (M), mimic control (MC), miR-582-5p inhibitor (I), and inhibitor control (IC) were synthesized by RiboBio (Guangzhou, China). Then, the HCMs were cultured in 6-well plates until the cell confluence reached about 80%. Next, the above shRNA, miR-582-5p inhibitor, or miR-582-5p mimic was transfected into the cells using Lipofect transfection reagent (CZ0002, Leagene Biotechnology) for 48 h. Finally, the cells were collected for OGD treatment or further experiments.