Opti mem 1 reduced medium

The miR-491-3p mimics (5′-AGTAGAAGGGAATCTTGCATAAG-3′) and its negative control (mimics NC) (5′-ACAGAGCTATAGATGTAAGTGAG-3′) were purchased from GenePharma Co., Ltd (Shanghai, China). miR-491-3p inhibitor (5′-CUUAUGCAAGAUUCCCUUCUAC-3′) and its negative control (inhibitor NC) ('5-GGUUCGUACGUACACUGUUCA-3′) were purchased from Sigma-Aldrich. Cells were transfected with miR-491-3p mimics, mimics NC, miR-491-3p inhibitor or inhibitor NC using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer instructions. Briefly, 5 μL Lipofectamine 2000 combined with miRNAs at a concentration of 30 nmol/L were incubated in Opti-MEM I reduced medium (Invitrogen) for 20 min prior to transfection. The mixture was then added into a 6-well plate with 5 × 10⁴ cells per well. Medium were changed to complete growth medium with 20% FBS after 6 h incubation at 37 °C. After transfection for 48 h, cells were harvested. Stannin (SNN) human tagged ORF clone lentiviral particle (oe-SNN), Lenti-ORF control particles (vector) and transfection reagent TurboFectin were obtained from OriGene and followed manufacturer’s preparation protocols.