Mir 92a inhibitor

The miR-92a mimics (5′-UAUUGCACUUGUCCCGGCCUGU-3′), mimics negative control (NC; 5′-CGGTGUGUUCAGACUACCUGUUC-3′), miR-92a inhibitor (5′-ACAGGCCGGGACAAGUGCAAUA-3′) and inhibitor NC (5′-TAACACGTCTATACGCCCA-3′) were obtained from Guangzhou RiboBio Co., Ltd. FBXW7 small interfering (si)RNA (si-FBXW7; sense, 5′-TAAAGAGTTGGCACTCTAT-3′ and antisense, 5′-ATAGAGTGCCAACTCTTTA-3′) and corresponding NC siRNA (si-scramble; sense, 5′-TTCTCCGAACGTGTCACGT-3′ and antisense, 5′-ACGTGACACGTTCGGAGAA-3′) were also purchased from Guangzhou RiboBio Co., Ltd.
Upon A549, H1299, H358 and NCI-H520 cells in six-well plates reaching 80% confluence, 1×10⁶ cells/well were transfected with the transfectants using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. A final concentration of 50 nM miR-92a mimics, 100 nM mimics NC, 200 nM miR-92a inhibitor, 100 nM inhibitor NC, 100 nM si-FBXW7 or 100 nM si-Scramble were used for each transfection. A blank control (untransfected cells) was set up for each transfection. Following transfection at 37°C for 24 h, the transfection efficiency was analyzed using RT-qPCR or western blotting.

The miR-92a mimic, negative control (NC) mimic, miR-92a inhibitor and NC inhibitor were purchased from Guangzhou RiboBio Co., Ltd., and were transfected into cells using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, cells were seeded in 6-well plates and transfected to ~80% confluence. The related mimic or inhibitor (50 nM) were transfected into cells using Lipofectamine 2000 in OPTI-MEM (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured for 6–8 h in an incubator at 37°C, then the transfection medium was discarded and cells were cultured with normal medium for 24 h at 37°C before use. The sequences of the miRNAs used were as follows: miR-92a mimic forward (F), 5′-UAUUGCACUUGUCCCGGCCUGU-3′ and reverse (R), 5′-AGGCCGGGACAAGUGCAAUAUU-3′; mimics NC F, 5′-UUCUCCGAACGUGUCACGUTT-3′ and R, 5′-ACGUGACACGUUCGGAGAATT-3′; miR-92a inhibitor, 5′-ACAGGCCGGGACAAGUGCAAU-3′; and miRNA inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA−3′.

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.