Mir 519 mimics

PD-L1 cDNA was subcloned and ligated into pCMV vectors. Lipofectamine 2000^® (Thermo Fisher Scientific, Inc.) was subsequently used to incorporate 2 µg pCMV-PD-L1 into 5×10⁵ PANC-1 and SW1990 cells. Next, ~48 h after transfection, the cells were harvested to perform subsequent experiments. Negative control (NC) mimic, NC inhibitor, miR-519 mimics or miR-519 inhibitors (Shanghai GenePharma Co., Ltd.) were transfected into PANC-1 or SW1990 cells using Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The sequences were as follows: NC mimic, 5′-GGUUCGUACGUACACUGUUCA-3′; miR-519 mimic, 5′-CUCUAGAGGGAAGCGCUUUCUG-3′; NC inhibitor, 5′-CCAUCAGUCCCAAAUCCA-3′; miR-519 inhibitor, 5′-CCAGAGGGAAGCGCCG-3′. NC mimic and NC inhibitor represented non-targeting sequences. At 36 h after transfection, the cells were subject to subsequent experiments.

To investigate the effects of miR-519 on cell viability, 5×10³ cells/well were seeded in 96-well plates in 100 µl RPMI-1640 supplemented with 10% FBS, and transfected with 50 nM miR-519 mimics or 50 nM negative control miR mimics (Genepharma Co., Ltd.) for 24, 48 and 72 h, as described above. MTT reagent (20 µl; Beijing Solarbio Science & Technology Co., Ltd.) was added to the wells 24 h post-transfection, prior to a 4-h incubation in darkness. The medium was then carefully discarded and blue formazan was dissolved with 200 µl dimethyl sulfoxide (Beijing Solarbio Science & Technology Co., Ltd.), and absorbance was measured at 550 nm (xMark, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Wells containing only HL60 cells served as controls.