Mir 590 5p inhibitor

Following 24 h of incubation at 37°C with 5% CO₂, NCI-H23 cells in the logarithmic growth phase were transfected with 500 pmol control small interfering RNA (siRNA) (cat. no. siN0000001-1-5; Ribobio), 500 pmol NUTM2A-AS1-siRNA (cat. no. siB180131045110-1-5; Ribobio), 50 nM inhibitor control (5′-GUCCAGUGAAUUCCCAG-3′; Shanghai GenePharma Co., Ltd.), 50 nM miR-590-5p inhibitor (5′-AAAUAUGCUGUAUGUCAUGUGUU-3′; Shanghai GenePharma Co., Ltd.), 100 nM mimic control (5′-CGCCAAUAUCAUUAUACCUC-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-590-5p mimic (5′-GAGCUUAUUCAUAAAAUGCAG−3′; Shanghai GenePharma Co., Ltd.), 1 µg control-plasmid (sc-437275; Santa Cruz), 1 µg METTL3-plasmid (sc-404029-ACT; Santa Cruz Biotechnology, Inc.), 500 pmol NUTM2A-AS1-siRNA + 50 nM inhibitor control, 500 pmol NUTM2A-AS1-siRNA + 50 nM miR-590-5p inhibitor, 100 nM miR-590-5p mimic + 1 µg control-plasmid or 100 nM miR-590-5p mimic + 1 µg METTL3-plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. 48 h after transfection, the transfection efficiencies were assessed using reverse transcription-quantitative PCR (RT-qPCR), and subsequent experiments were performed.

The miR-590-5p mimics, mimic control, miR-590-5p inhibitor, and inhibitor control were synthesized by GenePharma (Shanghai, People’s Republic of China). For cell transfection, cells were seeded in six-well plates (2×10⁵/well), and transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific). For luciferase activity analysis, the 3′-UTR of RECK containing the putative binding sites for miR-590-5p was amplified and cloned into the pGL3-luciferase reporter plasmid (Promega Corporation, Madison, WI, USA). Mutations in the miR-590-5p binding sites of RECK 3′-UTR were generated using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). For stably overexpressing miR-590-5p cells, the lentiviruses expressing miR-590-5p and control viruses were used to infect SGC-7901 cells.
The siRNA 5′-AAGACCCAGCCCUUGCCUCAA-3′ (sense) was used to knock down the expression of RECK, and the RNA 5′-AACGUUGCGAUAGCGUAGUAC-3′ was used as a control. The RECK coding region with and without the 3′-UTR was obtained from Hanbio (Shanghai, People’s Republic of China).