Mir 214 mimic

The Klf4 overexpression Klf4 plasmid was obtained from the scientific research group of Professor Keping Xie (University of Texas, MD Anderson Cancer Center, Houston, TX, USA). siKlf4 (forward primer, 5′-AUCGUUGAACUCCUCGGUCUCUCUC-3′; reverse primer, 5′-GAGAGAGACCGAGGAGUUCAACGAU-3′) was synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), as were miR-9-5p mimic (forward, 5′-UCUUUGGUUAUCUAGCUGUAUGA-3′; reverse, 5′-UUAGAAACCAAUAGAUCGACAUA-3′), miR-9-5p inhibitor (5′-UCAUACAGCUAGAUAACCAAAGA-3′), miR-9-5p inhibitor negative control (NC; 5′-CAGUACUUUUGUGUAGUACAA-3′), mimic NC (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′; reverse, 5′-ACGUGACACGUUCGGAGAATT-3′), miR-128 mimic (forward, 5′-UCACAGUGAACCGGUCUCUUU-3′; reverse, 5′-AGAGACCGGUUCACGGUGAUU-3′), miR-449a mimic (forward, 5′-UGGCAGUGUAUUGUUAGCUGGU-3′; reverse, 5′-CAGCUAACAAUACACUGCAAUU-3′) and miR-214 mimic (forward, 5′-ACAGCAGGCACAGACAGGCAG-3′; reverse, 5′-GCCUGUCUGUGCCUGCUGUUU-3′).

The miR-214 mimic (sense, 5′-aca gca gg cac aga cag gca gu-3′ and antisense, 5′-ugc cug ucu gug ccu gcu guu u-3′), mimic negative control (NC; sense, 5′-uuc ucc gaa cgu guc acg utt-3′ and antisense, 5′-acg uga cac guu cgg aga att-3′), miR-214 inhibitor (5′-ugc cug ucu gug ccu gcu guu u-3′) and inhibitor negative control (5′-cag uac uuu ugu gua gua caa-3′) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). PDLSCs (1×10⁶ cells/well) were plated in a six-well plate 1 day prior to transfection. PDLSCs were then transfected with miR-214 mimic, mimic NC, miR-214 inhibitor or inhibitor NC, at a final concentration of 25 nM, using siLentFect Lipid Reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, cells were cultured in serum-free Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 (Shanghai GeneChem Co., Ltd., Shanghai, China). Following 4 h of incubation at 37°C, the medium was replaced with DMEM/F12 supplemented with 10% FBS. The cells were harvested for further experiments at 48 h after transfection.

The two EBV⁺ NPC cell lines (C666-1 and HK-1) were obtained from the Cellbank of the Chinese Academy of Sciences. Cells were grown routinely in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and cultured in a 37°C humidified atmosphere with 5% CO₂. Ectopic expression of miR-214 in cells was achieved by transfection with miR-214 mimic (GenePharma, Shanghai, P.R. China) using Lipofectamine 3000 (Invitrogen). Overexpression of LINC0086 was achieved by using lentivirus containing LINC0086-expressed plasmid (GeneCopoecia, Guangzhou, P.R. China). Cells were plated into 6-well clusters or 96-well plates and transfected for 24 or 48 h. Transfected cells were used in further assays.