Cells
Jurkat cells were obtained from the Chinese Type Culture Collection Center of Wuhan University (Wuhan, China). They were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (both from Gibco BRL, Gaithersburg, MD, USA) in an incubator set to 37°C and 5% CO2 (Forma 3110 model; Thermo Fisher Scientific, Waltham, MA, USA). Actively growing cells were selected for seeding in 12-well culture plates (Corning Inc., Corning, NY, USA) at a density of 1.5×106 cells/well, and then were returned to the incubator under the same growth conditions. Fresh Opti-MEM culture medium (Gibco BRL) was added to cells before transfection.
According to the manufacturer's instructions, Opti-MEM culture medium was used to dilute miR-146a mimic and miR-146a inhibitor (both from Guangzhou Ruibo Biotechnology Co., Ltd., Guangzhou, China) and Trans-EZ transfection reagent (Shanghai Shengbo Biotech Co., Ltd., Shanghai, China). Trans-EZ transfection reagent and miR-146a mimic were sufficiently mixed with the inhibitor to form stable transfection complexes.
Electrotransfection of miR-146a overexpression vector
Cell Line Nucleofector® Solutions V electrotransfection reagent was mixed with supplement according to the proportion of 4.5:1. Cells (1×106) were centrifuged and resuspended in 100-µl electrotransfection reagent and allowed to incubate for 10 min. Two micrograms of miR-146a overexpression vector (Zimmer, Shanghai, China) encoding DNA, 2 µg negative control DNA vector, and 2 µg pmaxGFP® vector were added to the cell suspensions and mixed. The product was then transferred to an electrotransfection cup, and the cover was fastened down. An Amaxa electrotransfection instrument (Bio-Rad, Berkeley, CA, USA) was used to perform the electrotransfection procedure, and the program was started. Transfection efficiency of the cells was observed under a fluorescence microscope (AX-70, Olympus, Tokyo, Japan) and images were captured.
Experimental grouping
Jurkat cells were treated as follows for western blot analyses and FACS analysis of apoptosis: Control group (untreated Jurkat cells), empty vector group (Jurkat cell transfected with empty vector), agonist group (Jurkat cell transfected with miR-146a mimic) and inhibitor group (Jurkat cells transfected with miR-146a inhibitor).
Western blot analysis
Total protein was extracted and measured for purity according to a previous study (4). Protein samples were separated by SDS-PAGE electrophoresis (Bio-Rad mini vertical electrophoresis apparatus). Separated protein was then transferred to cellulose acetate membranes (NC membranes). Membranes were blocked in 5% skim milk in TBST. Membranes were then incubated in primary antibody [mouse anti-STAT1, p-STAT1 and Bcl-xL mAbs, used at a dilution of 1:1,000 and rabbit polyclonal β-actin antibody were all obtained from BIOSS (Beijing, China) and used at a dilution of 1:500]. Secondary antibody (horseradish peroxidase-labeled secondary antibody was purchased from Sigma (St. Louis, MO, USA) and used at a dilution of 1:200. ECL reagent was used for signal development (Amersham Biosciences, Uppsala, Sweden). Membranes were exposed and photographed with a FluorChem 8000 gel-imaging analyzer (Alpha Innotech, San Leandro, CA, USA).
Flow cytometry (FCM) (FACS)
We used an Annexin V/PI double-staining FCM reagent kit (C60 Biotech) and a flow cytometer (FACScalibur model; Becton-Dickinson, Franklin Lakes, NJ, USA) installed with CellQuest software to conduct the analysis.
Statistical analysis
SPSS 20.0 software (Chicago, IL, USA) was used to conduct statistical analyses. Data are expressed as mean ± standard deviation, and single-factor ANOVA analysis was used for comparison among groups. P<0.05 was considered to indicate a statistically significant difference.