Mir 181a mimic

ANRIL-overexpressing plasmids were constructed by inserting the ANRIL coding sequence into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). Oligonucleotides, including miR-181a mimic (5′-AACAUUCAACGCUGUCGGUGAGU-3′), negative control (NC) mimic (5′-UUCUCCGAACGUGUCACGUTT−3′), miR-181a antagomir (5′-ACUCACCGACAGCGUUGAAUGUU-3′) and NC antagomir (5′-CAGUACUUUUGUGUAGUACAA−3′), were purchased from Guangzhou RiboBio Co., Ltd. Small interfering (si)RNA targeting SIRT1 (siSIRT1, 5′-CCCUGUAAAGCUUUCAGAATT−3′) and NC siRNA (siNC; 5′-UUCUCCGAACGUGUCACGUTT−3′) were obtained from Shanghai GenePharma Co., Ltd. When the cell density reached 50–60% confluence, cell transfection was performed with Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Equal amounts of empty pcDNA3.1 vector (4 µg/well), NC mimic, NC antagomir (50 or 150 nM) or siNC (10 or 20 nM) diluted in the same volume of transfection reagents were transfected as controls, depending on the experimental purposes. The efficiency was examined 2 days after transfection. For H/R treatment, cells were cultured under normoxia or exposed to H/R (hypoxia for 4 h followed by reoxygenation for 8 h) at 2 days after transfection. The transfection efficiency for all transfections was effective under normoxic or H/R treatment conditions.

BCPAP cells or HUVECs were transfected with miR-181a mimic (100 pmol), miR-181a inhibitor or their corresponding negative controls (NCs; RiboBio, Guangzhou, China) by Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). In addition, HUVECs were also transfected with si-NC, si-MLL3, vector-NC, and MLL3-vector (Gene Pharma, Shanghai, China) by Lipofectamine 2000 (Invitrogen).⁴⁶