Negative sirna control

EBV-negative nasopharyngeal carcinoma (NPC) cell lines (5-8 F and HNE2), EBV-positive NPC cell line (C666-1) and EBV-negative gastric cancer (GC) cell line AGS were grown in RPMI 1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Grand Island, NY, USA). HEK293T cells were grown in Dulbecco's modified Eagle's medium. Cultures were maintained at 37 °C in a humidified environment with 5% CO₂. EBV miRNAs mimics and negative siRNA control were purchased from Qiagen Company (Valencia, CA, USA). LOC533103 siRNA was synthesized by Ruibo Company (Guangzhou, China). Hiperfect transfection reagent (Qiagen) was used for transfection of miRNA mimics or oligonucleotides for RNA interference (RNAi). The cDNA encoding lncRNA LOC553103 was PCR-amplified and subcloned into the BamH1 and EcoRI sites of the pcDNA3.1 vector (Invitrogen, Breda, The Netherlands), named pcDNA3.1-LOC553103 using Magic Cloning Biological Reagent (Light of Life Biotechnology Co., Ltd, Changsha, China). Plasmid transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.

Small interfering RNA (siRNA) designed against DAAM2 (M-014010-00-0005; Dharmacon, Lafayette, CA, USA) or a negative siRNA control (Qiagen, Valencia, CA, USA) were combined with lipofectamine (RNAiMax; Invitrogen) in optimem (ThermoFisher Scientific) to complex for 20 min at room temperature. After equilibration of the isolated cytotrophoblasts overnight (described above), fresh trophoblast media (DMEM, 10% FCS, no AA) was added to each well and siRNA complexes added in a dropwise manner. Cytotrophoblast cells with siRNA were cultured at 37 °C for 48 h under 8% O₂ (normoxic conditions) or 1% O₂ (hypoxia). Following this, media and cells were collected for subsequent analysis.

SH-SY5Y cells at ~80% of confluence were cotransfected with SURF4 siRNA from Ambion or a negative siRNA control from Qiagen (Hilden, Germany) with a pGFP reporter. RNAiMax transfection kit (Thermo Fisher technology, Waltham, MA, USA) was used to transfect cells in Opti-MEM. A total of 180 pM siRNA were added to cells plus 0.6 μg of pGFP reporter plasmid for cotransfection in 6-well plate and 72 pM siRNA plus 0.24 μg of pGFP in 24-well plate. Cells were transfected and incubated with growth media for 48–72 h.