Pcdna xist

PcDNA-XIST (OV-XIST group), sh-XIST (KD-XIST group), miR-124–3p mimic, miR-124–3p inhibitor, sh-IRF1 and the corresponding negative control were synthesized by Genepharma (Shanghai, China). The above-mentioned plasmids were separately transfected into BV2 cells through Lipofectamine 3000 (Invitrogen, CA, USA). After 48 h, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) or Western blot assay was utilized to assess the efficiency of transfection.

Melanoma cell lines (A375 (ATCC^® CRL-1619), SK-MEL-110 (ATCC^® HTB-67), HS-1 (ATCC^® CRL-9446), MEL-RM (ATCC^® HTB-70), and A2508 (ATCC^® CRL-11147)) and normal human epidermal melanocytes (HEMa-LP(ATCC^® PCS-200-013)) were obtained from American Type Culture Collection and cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Gibco-BRL, Grand Island, NY, USA) and Medium 254 (Cascade Biologics, Portland, OR, USA) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO), penicillin (100 μL/mL), streptomycin (100 mg/mL) and glutamine. All cells were maintained in a 37°C atmosphere that containing 5% CO₂. pcDNA-XIST (Rho-associated coiled-coil containing protein kinase 1, XIST-overexpressing plasmid), si-XIST (siRNA targeting XIST), si-ROCK1 (siRNA targeting ROCK1), si-NC (negative control siRNAs), miR-139-5p mimic (overexpressing oligonucleotides) and mimic NC (negative control) were all obtained from GenePharma (Shanghai, China). Lipofectamine 3000 (Invitrogen) was utilized for transfecting all oligonucleotides and plasmids into melanoma cells.