Nonspecific mimic control

The MiTarget microRNA 3′-UTR target vector (pEZX-MT01) containing full-length 3′-UTR of PTEN with two binding sites for EBV-miR-BART1-5p and EBV-miR-BART1-3p (wild-type 3′-UTR) was produced by GeneCopoeia. Two mutant sites (mut1 and mut2) in PTEN 3′-UTR were then generated by site-directed mutagenesis with KOD-Plus-Mutagenesis Kit (SMK-101, Toyobo C. Ltd). For luciferase reporter assays, wt or mut 3′-UTR vector was co-transfected with EBV-miR-BART1 mimic or nonspecific mimic control (Genepharma) into HEK 293 T cells, respectively. Luciferase activity was measured 48 h after transfection using Luc-Pair miR Luciferase Assay Kit (GeneCopoeia) on Panomics Luminometer.

HeLa and CaSki cell lines purchased from American Type Culture Collection (Manassas, VA, USA) and the cell bank of the Chinese Academy of Science (Shanghai, China), respectively, were cultured at 37°C in 5% CO₂ in a humidified incubator in Dulbecco's modified Eagle's medium and RPMI 1640 medium, respectively. All cells were authenticated by short tandem repeat profiling before receipt and were propagated for less than 6 months after resuscitation.
miRNAs were transfected at a working concentration of 100 nmol/L using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). miR-187 mimics, a nonspecific mimic control, miR-187 inhibitor, and a nonspecific inhibitor control were all purchased from GenePharma (Shanghai, China). Protein and RNA samples were extracted from subconfluent cells during the exponential growth phase.