Cell lines and cell culture
Prostate cancer cell lines (DU145, PC3) and human NK cell line NK92 cell were obtained from the Chinese Academy of Sciences (Wuhan, China). Prostate cancer cell lineswere cultured in RPMI 1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 100 mg/mL penicillin/streptomycin. NK92 cells were maintained in RPMI 1640 supplemented with 10% human FBS and 100 U/ml recombinant human IL-2 (Peprotech, Rocky Hill, NJ). All cells lines were maintained at 37ºC in a humidified atmosphere containing 5% CO2.
Cell transfection and pterostilbene treatment
For miRNA transfection, prostate cancer cells were plated in six well plates and after growingup to 70–90% confluence, the cells were transfected with the synthesized miR-20a mimics or scramble control (Guangzhou Ribobio Co, Guangzhou, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. For pterostilbene treatment, cells were cultured with adding of pterostilbene (Cayman Chemical, Michigan, USA) dissolved in DMSO (Sigma, Saint Louis, USA) at a final concentration of 50μmol/L in medium. Cell samples were collected at 24 h after transfection or treatment with pterostilbene for further analysis.
qRT-PCR
Cell samples were lysed in RNAiso (TaKaRa, Dalian, China) for miRNA extraction, and miRNAs was reverse-transcribed into complementary DNAs (cDNAs) using PrimeScript Reverse Transcriptase (TaKaRa, Dalian, China) to produce a template suitable for qRT-PCR. U6 was used as an internal control. qRT-PCR was performed using SYBR Green master mix kit (Exiqon, Vedbaek, Denmark). This experiment was repeated 3 times and data were analyzed with the 2-∆∆Ct.
Luciferase reporter assay
TargetScanHuman 7.1 (www.targetscan.org) predicted that MICA/B was a potential target ofmiR-20a, but not the TGF-β1. To generate the luciferase reporter vectors, MICA/B 3’UTR segments (MICA/B 3’UTR WT and MICA/B 3’UTR MUT) were amplified by PCR from the human cDNA and inserted into the pmiR-RB-Report™ vector (Guangzhou RiboBio Co., Ltd., China). Then MICA/B 3’UTR luciferase construct and miR-20a mimics were co-transfected into DU145 and PC3 cells using Lipofectamine2000 transfection reagent (Invitrogen, Shanghai, China) according to manufacturer's instructions. After 48 h transfection, relative luciferase activity of MICA/B 3’UTR was measured by the Dual-Luciferase Reporter Assay (Promega, Madison, USA).
Analysis of NK cell degranulation
NK92 cells were co-cultured with PC3 or DU145 cells at a 3 : 1 ratio for 4 h. PE-anti-107a or isotype control monoclonal antibody (BD Biosciences, NJ, USA)and Golgi-Plug and Golgi-Stop eagents (BD Biosciences, NJ, USA) at 1:1500 dilution were added to the co-culture system at the beginning of the assay. Afterward, the cells were collected for being analyzed by flow cytometry.
Flow cytometry
For cell-surface protein expression analysis, cells were harvested, and incubated with antibodies for 30 min at 4°C in the dark. These antibodies are: APC-conjugated CD3, APC-cy5-conjugated CD56, PE-conjugated anti-NKG2D, FITC-conjugated anti-MICA, PE-conjugated anti-MICB (BD Biosciencesn, NJ, USA). For intracellular staining, cells were fixated and permeabilizated using BD Perm/Fix kits following surface markers staining and then stained withPE/Cy7-anti-IFN-γ (Biolegend, CA, USA). Before being collected, cells were cultured in the presence of Golgi-Plug and Golgi-Stop eagents at 1:1500 during the last 4 h. Isotype-matched monoclonal antibodies were used as controls.Cells were analyzed by flow cytometer (BD, NJ, USA). Data were analyzed using the FlowJo software (BD, NJ, USA).
Enzyme-linked immunosorbent assay
Culture supernatants were harvested and analyzed forTGF-β1production by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, CA, USA), following the manufacturer’s instructions.
NK cell cytotoxicity
NK cells cytotoxic to prostate cancer cells was analyzed using the CytoTox 96 cytotoxicity assay kit (Promega, Madison, USA) according to the manufacturer’s instructions. In brief, 1 × 104 DU145 or PC3 cells were seeded in a round-bottom 96-well plate as target cells. NK92 cells were then added as effector cells at E:T ratios of 10:1, 3:1 and 1:1. After 4 h of co-incubation, the supernatant was removed for analysis. The cytotoxicity of the effector cells against the target cells was assessed as: cytotoxicity = (experimental release - effector spontaneous release - target spontaneous release)/(target maximum release - target spontaneous release) × 100%.
Statistical analysis
Statistical analyses were performed using SPSS version 13.0. Values were expressed as the mean ± standard deviation. Statistically significant differences between groups in each assay were determined by ANOVA, or student's t-test. P< 0.05 was considered statistically significant.