Agomir 26a

PTEN 3′-untranslated region (UTR) sequences with wild-type and mutant seed regions for miR-26a were synthesized by Sangon, Shanghai, China, with the addition of Spe-1 and HindIII restriction sites at both ends. These two DNA fragments were cloned into the pMIR-REPORT luciferase reporter plasmid (E1980; Promega, Madison, WI, USA), and 0.8 µg plasmids carrying the wild-type and mutant 3′-UTR sequences, respectively, were transfected into 293T cells (Cell Bank, Chinese Academy of Sciences, Shanghai, China), using liposomes, followed by transfection with 100 nM agomiR-26a (Sangon Biotech). After 24 hours, the cells were lysed and luciferase was determined using a GloMax 20/20 luminometer (Promega), with Renilla luciferase as an internal reference.

For cell transfection, Penl1 PSCC cells (Cell Bank, Chinese Academy of Sciences) in logarithmic growth phase were inoculated onto a 24-well plate at a density of 3 × 10⁵ cells/well and cultured with Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), without antibiotics. The cells were transfected when they reached 70% confluence. Plasmid/small interfering RNA/agomiR 1 µL and Lipofectamine 2000 1 µL were added into 50 µl Opti Memi medium (Thermo Fisher Scientific) separate Eppendorf tubes (Thermo Fisher Scientific) for 5 minutes. The solutions in the two tubes were then mixed together for 20 minutes, and the mixture was incubated with the cells for 6 hours. The cultured medium was then replaced with DMEM/F12 containing 10% fetal bovine serum for another 48 hours before further analysis. The agomiR-26a and agomiR-NC (Sangon Biotech) sequences were as follows: agomiR-26a, forward 5′-UUCAAGUAAUCCAGGAUAGGCU-3′ and reverse 3′-AAGUUCAUUAGGUCCUAUCCGA-5′; and agomiR-NC, forward 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse 3′-TTAAGAGGCUUGCACAGUGCA-5′.