Anti mir 205

Primary mGCs were isolated as described based on our previous study [21 (link)]. mGCs were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12; Sigma-Aldrich, CA) supplemented with 10% inactivated fetal bovine serum (FBS; Gibco BRL, CA), penicillin (100 U/mL), and streptomycin (100 U/mL) (Life Technologies, CA) at 37°C in a humidified atmosphere with 5% CO₂. The media were changed every 48 h. The 2- or 3-passage mGCs were selected for subsequent experiments.
A siRNA directly targeting MALAT1 (si-MALAT1; 5′-GTGAATGAGTGATAAGTAA-3′) and appropriate nontargeting siRNA (si-NC; 5′-TTCTCCGAACGTGTCACGT-3′) were bought from Geneseed Biotech Co. (Guangzhou, China). In addition, GenePharma Co. Ltd. (Shanghai, China) provided miR-205 mimics (5′-UCCUUCAUUCCACCGGAGUCUG-3′), negative control mimics (miR-NC; 5′-GGUCCGUCCGUAAUUAUCCUCC-3′), and a miR-205 inhibitor (anti-miR-205; 5′-CAGACUCCGGUGGAAUGAAGGA-3′) as well as its negative control (anti-miR-NC; 5′-CAGACUCCGGUGGAAUGAAGGA-3′). The CREB1 overexpression plasmid came from our lab. mGCs (5 × 10³ cells/well) underwent transient transfection with the abovementioned constructs (100 nM siRNAs, mimics and inhibitors) using Lipofectamine 3000 (Invitrogen, CA, USA) based on provided protocols, with the efficiency of transfection being assessed at 48 h following transfection by quantitative real-time PCR (qRT-PCR).

Including paraffin‐embedded primary MM (62 cases), and normal skin tissues (20 cases) was purchased from Auragene Co. Ltd (cat No. TC0229; Changsha, China) for in situ hybridization confirmation of NORAD expression. Human melanocytes (HM) and four human MM cell lines (A375, WM451, SK‐MEL‐24, and WM35) purchased from the American Type Culture Collection (Manassas, VA) were cultured in RPMI‐1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) in a humidified atmosphere of 5% CO₂ at 37°C. Ectopic miR‐205 expression was observed after transfection with either pre‐miR‐205 or anti‐miR‐205 (Genepharma, Shanghai, China) using Lipofectamine 3000 (Invitrogen). Knockdown of NORAD was performed using lentivirus (Lv) containing short hairpin (sh)RNA sequences of NORAD (GeneCopoecia, Guangzhou, China). Cells were cultured in either 6‐well clusters or 96‐well plates for 24 or 48 hours, and then used to experiments or RNA/protein extraction.