Mir 92a mimic

Cells were seeded at a density of 6×10⁵ cells in a 6-well plate and grown to 60–80% confluency for transfection using Lipofectamine^®3000 reagent (Invitrogen, Carlsbad, CA, USA). An miR-92a inhibitor (Ribobio, Guangzhou, China) was used to transfect PC-3 cells to downregulate the expression of miR-92a. An miR-92a mimic (Ribobio, Guangzhou, China) was used to transfect LNCaP cells to overexpress the miR-92a. A SERTAD3 expression vector (FengHuiShengWu, Hunan, China) was transfected into PC-3 to confirm the dependency of target genes on miR-92a. MicroRNA mimic, inhibitor or SERTAD3 cDNA were incubated in 125 µL of Opti-MEM ^® (Gibco, Grand Island, NY, USA) medium for 15 min at room temperature (RT), respectively. Next, 5 µL of Lipofectamine was added to 125 µL of Opti-MEM^® medium mixture for 15 min at RT to prepare the transfection solution. The cells were maintained in 1.5 mL of DMEM including the transfection solution for 12 h at 37°C. Then, the solution was removed and replaced with fresh serum-supplemented medium and cultured for 24 h. The transfected cells were harvested to determine the transfection efficiency by qRT-PCR. In this study, a randomly scrambled sequence of mimic or of inhibitor (Ribobio, China) served as the negative control (NC) for non-sequence-specific effects in miRNA experiments.

The miR-92a mimic, inhibitor, and negative control miRNA were purchased from RiboBio (Guangzhou China) and transfected into cells at 100 nM concentrations via Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. KLF4 plasmid (1 µg; Origene, Rockville, MD, USA) was also transfected into cells in the presence or absence of the miR-92a mimic by Lipofectamine 2000. After transfection twice in 48 h, cells were used in the subsequent experiments. RT-PCR and Western blotting were used to evaluate the transfection efficacy.

Rat aortic endothelial cells were obtained from American Type Culture Collection (ATCC) and maintained in DMED cell culture medium supplemented with 10% fetal bovine serum in a 37℃ cell culture incubator with 5% CO₂. The miR-92a mimic and miR-92a inhibitor were from RiboBio. The transfection was conducted using the complexes of Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) and miR-92a inhibitor (100 nM)/mimic (50nM) or negative controls according to the manufacturer’s instructions. Dorsomorphin (compound C) was obtained from SelleckChem (Houston, TX, USA).