Cell proliferation assay and flow cytometry-based apoptosis analysis

CCK-8 assay was used to study cell proliferation. Cells were seeded in 96-well plates at the density of 1 × 10³ (cells/well), and the absorbance at 450 nm was measured on 24, 48, 72, 96, 120 h with 10 μL of CCK-8 solution. Proliferative comparison of mimic-transfected cells or inhibitor-transfected cells was calculated by normalization with respect to NC: % cells = (mimic or inhibitor – corresponding NC)/ corresponding NC × 100. Difference of OD Value on 120 h and OD Value on 24 h was used for calculation. Each assay was carried out in triplicate.

Flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Invitrogen) was used to measure cell apoptosis. Cells were seeded in 6-well plates and digested after 48 h. Then, 1X Binding Buffer was used to resuspend cells. We stained cells with 5 μL of Annexin V-FITC Conjugate and 5 μL of Propidium Iodide Solution and incubated them for 30 min. Stained cells were analyzed using a FACScalibur (Becton-Dickinson). For comparison, % apoptotic cells = (mimic or inhibitor – corresponding NC)/ corresponding NC × 100. Experiments were conducted in triplicate.