Mir nc mimic

miR-NC mimics and miR-106a mimics were purchased from Shanghai GenePharma Co., Ltd. The sequences were: miR-NC mimics, 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-106a mimics, 5′-AAAAGUGCUUACAGUGCAGGUAG-3′. miR-NC mimics or miR-106a mimics were transfected at a final concentration of 40 nM with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. In brief, miR-NC mimics or miR-106a mimics and Lipofectamine^® 2000 were mixed (to a concentration of 200 nM) in Opti-MEM medium (Thermo Fisher Scientific, Inc.) and incubated for 20 min at 37°C. Subsequently, the mixture was added to HCT116 and HT-29 cells in DMEM. The medium was replaced with fresh DMEM after 6 h and sustained for 18 h before subjected to following experiments.

The human PCa cell line LNCaP was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and routinely cultured in RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂.
miR-106 inhibitors, miR-negative control (NC) inhibitor, miR-106 mimics and miR-NC mimics were purchased from Ambion (Thermo Fisher Scientific, Inc.). Inhibitors and mimics were transfected at a concentration of 100 nM. LNCaP cells were seeded into 6-well plates and transfected with 100 nM of miR-106 inhibitor, miR-NC inhibitor, miR-106 mimics and miR-NC mimics using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols for 48 h. The sequences of miR-106 inhibitor, miR-NC inhibitor, miR-106 mimics and miR-NC mimics were as follows: miR-106b mimics, 5′-TAAAGTGCTGACAGTGCAGAT-3′; miR-NC mimics, 5′-UUCUCCGAACGUGUCACGUTT-3′; miR-106b inhibitor, 5′-AUCUGCUCAGCACUUUA-3′; miR-NC inhibitor, 5′-CAGUACUUUUGUGUAGUACAA-3′.

MiR-218-5p mimics, miR-218-5p inhibitors, short hairpin RNA (shRNA) targeting DEK (sh-DEK), pcDNA 3.1-DEK plasmid (pcDNA3.1/DEK), and controls (miR-NC mimics, miR-NC inhibitors, sh-control, pcDNA 3.1 plasmid) were all obtained from Thermo Fisher Scientific. According to the manufacturer’s instructions, Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) kit was used for transfection. The sequences were shown as follows: sh-DEK (sense: 5ʹ-GGAGAGATCAGGTGTAAATAG-3ʹ, anti-sense: 3ʹ-CTATTTACACCTGATCTCTCC-5ʹ).

TargetScan (www.targetscan.org) software predicted that SNHG12 was a potential target gene of miR-16. SW620 and HT-29 cells were transfected with a wild-type (WT) or a mutant (MT) SNHG12 pMIR-REPORT™ miRNA Expression Reporter Vector (Ambion; Thermo Fisher Scientific, Inc.) containing a 4-bp mutation at the predicted miR-16 binding site within SNHG12, along with miR-16 mimics (cat. no. 4464066; Thermo Fisher Scientific, Inc.) or miR-NC mimics (cat. no. 4464058; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000. After transfection for 48 h, a luciferase reporter gene assay was performed using the Dual-Luciferase^® Reporter Assay System (Promega Corporation). The activity of firefly luciferase was normalized to the activity of Renilla luciferase.

miR-NC inhibitor, miR-620 inhibitor, miR-NC mimic and miR-620 mimic were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows: miR-NC inhibitor, 5′-UUCUCCGAACGUGUCACGUUU-3′; miR-620 inhibitor, 5′-AUUUCUAUAUCUCCAUUU-3′; miR-NC mimic, 5′-UCGCUUGGUGCAGGUCGGG-3′; miR-620 mimic, 5′-AUGGAGAUAGAUAUAGAAAUUU-3′. The control group was treated with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 30 nM miR-NC inhibitor, miR-620 inhibitor, miR-NC mimic and miR-620 mimic were transfected with Lipofectamine 2000 into cells at 37°C 24 h prior to subsequent experiments.

Transformed Human Liver Epithelial cell line (THLE-2) and the human hepatocellular carcinoma cell line (Huh-7, Hep3B) were bought from ATCC (Rockville, MD). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen; Thermo Fisher Scientific) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific). The cells were cultured at 37°C in a moist atmosphere with 5% CO₂.
si-NC (Negative Control), si-SNHG14-1, si-SNHG14-2, miR-217-5p mimic, miR-NC mimic, miR-217-5p inhibitor and miR-NC inhibitor were synthesized by GenePharma (Suzhou, China). For silencing of SNHG14, si-NC or si-SNHG14-1 or si-SNHG14-2 were transfected into indicated cells with Lipofectamine RNAiMax (Invitrogen; Thermo Fisher Scientific) following manufacturer’s protocol. MiR-217 mimic, miR-NC mimic, miR-217 inhibitor or miR-NC inhibitor were transfected into cells with Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific). At 48 hours after transfection, cells were subjected to RNA extraction and RT-qPCR to validate transfection efficiency. The sequence: si-NC:5ʹ-CCAUGAGGUCAUGGUCUG-3ʹ; si-SNHG14-1:5ʹ-GAGAUGGAUCAACAGUAU-3ʹ; si-SNHG14-2:5ʹ-GCUACAAUCACUAUGAAUC-3ʹ; miR-217 mimic:5ʹ-UACUGCAUCAGGAACUGAUUGGA-3ʹ; miR-NC-mimic:5ʹ-UUGUCCGAACGUGUCACGU-3ʹ; miR-217 inhibitor:5ʹ-UCCAAUCAGUUCCUGAUGCAGUA-3ʹ; miR-NC inhibitor:5ʹ- CAGCUGGUUGAAGGGGACCAAA-3ʹ.