Construction of pGPU6/GFP/Neo-shRNA carrier
The plasmid pGPU6/GFP/Neo was purchased from GenePharma and the negative control plasmid (no homology to disrupt the sequence) was provided by GenePharma. We designed and synthesized four pairs of inference shRNA oligos (Shanghai Invitrogen Biotechnology Co., Ltd, China). Annealed shRNA oligos were ligated into the pGPU6/GFP/neo vector before transformation into E. coli. We selected 3 clones from each plate for growth in LB liquid medium containing 50 mg/L kanamycin overnight at 37 °C. Clones were analyzed by sequencing (Shanghai Invitrogen Biotechnology Co., Ltd, China). The four target sites for PIM1 shRNA are shown below:
Transfection of ACC-M cells
ACC-M was purchased from the Cell Bank for Type Culture Collection, Chinese Academy of Sciences (Beijing, China), and cultured in RPMI-1640 medium (Gibco) supplemented with fetal calf serum (10% Hyclone), penicillin (100 U/mL) and streptomycin (100 μg/mL). ACC-M cells were seeded in a 6-well culture plate at a density of 1 × 105 cells/well. After 24 h, transfection complexes were assembled as follows: 4 μg of pGPU6/GFP/Neo-shRNA plasmid or negative control plasmid was resolved in 400 μL RPMI 1640 medium (serum-free and antibiotic-free). To this, 9 μL Lipofectamine™ 2000 transfection reagent was added, thoroughly mixed, and allowed to stand at room temperature for 10 min to form transfection complexes. The transfection mix was added to wells and cells were cultured at 37 °C in a 5% CO2 incubator. The cells were collected 24–48 h after transfection.
Reverse transcriptase (RT)-PCR and quantitative real-time PCR (qRT-PCR)
RT-PCR and qRT-PCR were performed as described previously . The following primers were used:
PIM1 forward, 5′-TGTGCTGGGAGAAATACTTGAA-3′ and reverse, 5′-AGGTGGCTCAGCGTTTGG-3′ (size: 160 bp);
p21 forward, 5′-GGAGACTCTCAGGGTCGAAAACG-3′ and reverse, 5′-GAGAAGATCAGCCGGCGTTTG-3′ (size: 77 bp);
RUNX3 forward, 5′-CCAAGGCACCTCGGAACTGAAC-3′ and reverse, 5′-TCTCCGTGAGGGTTGGCAGC-3′ (size: 83 bp);
GAPDH forward, 5′-CATGAGAAGTATGACAACAGCCT-3′ and reverse, 5′-AGTCCTTCCACGATACCAAAGT-3′ (size: 113 bp).
After PCR amplification, dissociation of SYBR Green-labeled cDNA (melt curve analysis) was carried out to affirm that there were no nonspecific PCR products. 2−∆∆Ct method was performed to analyze the relative quantification of transcript expression.
Detection of protein expression by western blot
Cells were harvested at 80% confluence and lysed by suspension in RIPA lysis buffer followed by gentle sonication. Protein lysates (20 μg) were separated on 8% SDS polyacrylamide gel by electrophoresis and transferred to PVDF membrane (Millipore). Membranes were then blocked in PBS/Tween (PBS with 5% non-fat powered milk and 0.05% Tween 20) at room temperature for 2 h. The blot was then incubated with affinity-purified rabbit anti-PIM1 (Novus NBP1-40501, 1:500) overnight. The membranes were washed 3–4 times with PBS/Tween and incubated with HRP-conjugated goat anti-rabbit IgG (JIR 111-035-003, 1:8000) for 2 h at room temperature. After repeating the washing steps, the signal was detected by chemiluminescence using the Pierce Super Signal West Pico Systems (Pierce).
Cell cycle analysis
ACC-M cells (1 × 106/mL) in logarithmic growth phase were plated in 6-well plates and transfected as described above. The cells were fixed with ice-cold 70% ethanol at − 20 °C for 12–24 h. The cells were collected after centrifugation and resuspended in 500 μL of PBS buffer containing RNase A (the final concentration of RNase A was 0.25 mg/mL) and incubated at 37 °C for 30 min. Five microliter PI was added and incubated for 30 min at room temperature in dark. Data were acquired by flow cytometry (FACS Calibur, Becton–Dickinson).
Immunofluorescence staining analysis
ACC-M cells were transfected as described. After 48 h, cells were washed twice with PBS. Following fixation with 4% paraformaldehyde (15 min, 37 °C), cells were washed three times in PBS and then permeabilized with 0.1% Triton X-100 in PBS for 2 min at room temperature. Samples were blocked with 3% BSA and 0.05% Tween 20 in PBS (blocking solution) for 30 min at room temperature and then incubated overnight at 4 °C with primary antibodies (PIM1, Novus NBP1-40501, 1:200; p21, Novus NBP200-303, 1:200; RUNX3, Novus NBP1-46694, 1:200), respectively. For the immunofluorescence staining method, cells were incubated with secondary antibodies (1 µg/mL), DyLight 488 AffiniPure Goat Anti-Mouse IgG (H+L) (EarthOx) at 1:100 for Novus NB200-303 and DyLight 594 AffiniPure Goat Anti-Rabbit IgG (H+L) (EarthOx) at 1:100 for Novus NBP1-40501 and Novus NBP1-46694, There secondary antibodies were conjugated to Alexa-488 green fluorescence (Molecular Probes, Eugene, OR). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Samples were visualized by confocal microscopy at 63× magnification.
Ninety-seven patients with histopathologically proven SACC in Zhejiang Cancer Hospital between July 2002 and July 2014 were recruited for this study. The study was approved by the Ethics Committee of Zhejiang Cancer Hospital. All patients have signed the informed consent.
Paraffin-embedded tissues were cut for 4 μm sections mounted on glass slides (Mats-unami,MS-coated glass) and dried overnight at 37 °C. After deparaffinization, antigen retrieval in 0.01 M citrate buffer, and inactivation of endogenous peroxidase activity in 3% H2O2/methanol, the slides were incubated with antibody for PIM1 (Novus NBP1-40501, 1:200) and RUNX3 (Novus NBP1-46694, 1:200) at 4 °C overnight. The immunoreactivity was visualized by using a streptavidin–biotin–peroxidase staining kit (Nichirei, Histofine Simple Stain Max PO Multi) and DAB solution (Nichirei, Simple Stain DAB). The results were presented as percentage of nucleus staining positive cells relative to total cells. The scores of staining results were given as negative and positive. In brief, IHC score was determined by combining staining frequency and intensity. In detail, the staining frequency score was defined as no cell stained scored as 0, 1–10% of cells stained as 1, 11–50% of cells stained as 2, 51–80% of cell stained as 3, and 81–100% of cell stained as 4. Staining intensity score was rated on a scale of 0–3, with 0 for negative; 1 for weak; 2 for moderate; and 3 for strong staining. Theoretically, the scores could range from 0 to 12. An IHC score of 9–12 was considered as strong immunoreactivity, 5–8 as moderate, 1–4 as weak and 0 as completely negative. Sections in which the staining could not be well characterized were considered as equivocal. Staining was scored independently by two pathologists who were blinded to each other’s findings. All conflicting calls on scoring were adjudicated by a third individual. We used IHC score 5–12 for normal to high expression (positive) and 0–4 for no to low expression (negative).
SPSS 22.0 was used to perform one-way ANOVA for the discrepancy of three groups or more. The two-tailed Student’s t-test was used to detect any statistically significant difference between two groups. Associations between PIM1, RUNX3 levels, and clinicopathologic parameters were analyzed by using the χ2 test or the Fisher exact test. Survival analysis was carried out by the Kaplan–Meier method and significant differences were assessed by means of the log-rank test. p values < 0.05 were considered to be statistically significant.