Mir 221 inhibitor

Jurkat cells were seeded into 6-well plates (1x10⁶ cells/well) and cultured at 37˚C for 24 h. Then, cells were transfected with 100 nM inhibitor control (the negative control of miR-221 inhibitor; 5'-CAGUACUUUUGUGUAGUACAA-3'; Guangzhou Ribobio Co., Ltd.), 100 nM miR-221 inhibitor (miR-221 antagonist' 5'-GAAACCCAGCAGACAAUGUAGCU-3'; Guangzhou Ribobio Co., Ltd.), 50 nM mimic control (the negative control of miR-221 mimic; 5'-CGGUACGAUCGCGGCGGGAUAUC-3'; Guangzhou Ribobio Co., Ltd.), 50 nM miR-221 mimic (miR-221 agonist; 5'-AGCUACAUUGUCUGCUGGGUUUC-3'; Guangzhou Ribobio Co., Ltd.), 100 nM miR-221 inhibitor + 0.2 µM control-small interfering (si)RNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.) or 100 nM miR-221 inhibitor + 0.2 µM PTEN-siRNA (cat. no. sc-29459; Santa Cruz Biotechnology, Inc.) using Lipofectamine 3000 reagent, according to the manufacturer's instructions. The transfection efficiency was detected 48 h later using RT-qPCR.

The miR-221 mimics, miR-221 inhibitor and scrambled sequence pre-miR negative control (NC) were purchased from a commercial manufacturer (Guangzhou RiboBio, Co., Ltd., Guangzhou, China). Ovarian cancer cells (100 µl) were seeded in 24-well plates (1×10⁶ cells/ml) and incubated for 24 h, then cells were transfected with miRNA mimics/inhibitor (50 mM) or vectors (2 µg) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in serum-free medium in accordance with the manufacturer's instructions.

LX2 cells were used for cell transfection. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was diluted with Optimem I (Gibco; Thermo Fisher Scientific, Inc.) to a selected optimal concentration and then transfected into LX2 cells. LX2 cells were well cultured until 60–70% confluence. LX2 cells (1×10⁵) were transfected with 50 nM miR221 mimics (5′-ACCUGGCAUACAAUGUAGAUUU-3′), 100 nM miR221 inhibitor (5′-AAAUCUACAUUGUAUGCCAGGU-3′) and their matched negative controls (25 nM NC-miR mimic, 5′-UUCUCCGAACGUGUCACGUTT-3′ and 25 nM NC-miR inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′) using Lipofectamine^® 2000 in a 37°C incubator with 5% CO₂. The cells were collected 48 h after transfection. miR221 mimics, miR-NCs and miR221 inhibitor were purchased from Guangzhou RiboBio Co., Ltd. Furthermore, LAMP2 overexpression plasmids (LAMP2 group; Shanghai GeneChem Co., Ltd.) or empty pcDNA3.1 plasmids (NC group; Shanghai GeneChem Co., Ltd.) were transfected into LX2 cells at a multiplicity of infection (MOI) of 50, according to the manufacturer's instructions. The cells were transfected for 48 h at 37°C with 5% CO₂.