Mir nc inhibitor

miR-665 inhibitor (5′-AGGGGCCUCAGCCUCCUGGU-3′), miR-665 mimic (5′-ACCAGGAGGCUGAGGCCCCU-3′) and the corresponding negative controls (miR-NC; 5′-UCGCUUGGUGCAGGUCGGGAA-3′) were synthesized and purchased from Shanghai GenePharma Co., Ltd. SKOV3 and ES2 cells were seeded into each well of 24-well plates (2×10⁵ cells per well) and were transfected with miR-665 inhibitor, miR-665 mimic, miR-NC inhibitor or miR-NC mimic at a concentration of 50 nM with Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol, and maintained for 48 h before any subsequent experiments were performed.

miR-615-3p and its positive control (miR-NC), as well as miR-615-3p inhibitor and its negative control (miR-NC inhibitor), were purchased from Ambion (GeneCopoeia, Guangzhou, P.R. China). Transfection of miR-615-3p or inhibitor was carried out using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad CA, USA) according to the manufacturer’s protocol.

miR-330-5p mimics, miR-330-5p inhibitor, and their controls (miR-NC or miR-NC inhibitor) were purchased from Ambion (GeneCopoeia, Guangzhou, China). lentiviral vector encoding WT1-AS cDNA (LV-WT1-AS) was constructed by Genepharma Co., Ltd (Shanghai, China). shRNA-WT1-AS was chemically synthesized by Realgene (Nanjing, Jiangsu, China). The shRNA targeting p53 was designed by Genepharma Co., Ltd. The lentiviral vector encoding p53 cDNA was constructed by Genepharma Co., Ltd, and named as LV-p53. The empty vector was control (named as LV-vector). In brief, cells were maintained around 50–60% confluence in 10 cm culture dishes when transfected with plasmids. Transfection of WT1-AS or miRNAs was conducted using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. To establish stable cell lines, media were refreshed on transfected cells 4 days later with media containing puromycin (1.5 µg/mL). Transfected cells were treated with puromycin, refreshed every 3 days, for 2 weeks.

miR-205-5p mimic, miR-negative control (NC) mimic, miR-205-5p inhibitor and miR-NC inhibitor were purchased from Shanghai GenePharma Co., Ltd. The sequences were: miR-205-5p mimic, 5′-UCCUUCAUUCCACCGGAGUCUG-3′; miR-NC mimic, 5′-UCGCUUGGUGCAGGUCGGGAA-3′; miR-205-5p inhibitor, 5′-CAGACUCCGGUGGAAUGAAGGA-3′; miR-NC inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′. A total of 50 nM miRNA was transfected into MDA-MB-231 and BT549 cells using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) according to manufacturer's protocol for 48 h at 37°C. Cells were then subjected to the following experiments.

Cells were treated with 10 µg/ml of cisplatin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). SW480 cells in the logarithmic stage were were transfected with 200 nmol/l miR-1271 mimic, miR-NC mimic, 200 nmol/l miR-1271 inhibitor or miR-NC inhibitor (all Thermo Fisher Scientific, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Following 72-h transfection, the cisplatin-miR-1271 mimic, cisplatin-miR-1271 inhibitor and the cisplatin group were treated with cisplatin (10 µg/ml) for 48 h at 37°C. The cells of the control group were untreated. The sequences of miR-1271 mimics and miR-NC mimics were as follows: 5′-CUUGGCACCUAGCAAGCACUCA-3′ and 3′-UUCUCCGAACGUGUCACGUTT-5′. The sequences of miR-1271 inhibitor and miR-NC inhibitor were 5′-UGAGUGCUUGCUAGGUGCCAAG-3′ and 5′-UUGUACUACACAAAAGUACUG-3′, respectively.