Immunohistochemical staining
For immunohistochemical staining, thirty-six surgically resected human cervical carcinoma tissue samples were collected from the Department of Obstetrics and Gynecology, Wuhan Union Hospital. The study was approved by the institutional review boards. Immunohistochemical staining was performed according to our previous protocol [14]. Briefly, human tumor tissues were embedded in paraffin and cut into 5-μm sections that were placed onto glass slides. After antigen retrieval, sections were stained for the expression of PLK-1 (BD Biosciences, San Diego, CA) (1:100)detected by streptavidin-biotin-horseradish peroxidase complex formation. Tumor sections stained for IgG instead of primary antibodies were used as the negative control. The immunoactivities of PLK-1 were ranked according to the percentage of positive tumor cells: score 3 (> 75%), score 2 (25-75%), score 1 (< 25%), and score 0 (negative).
Cell culture, transient transfection, RNA interference, and cisplatin treatment
HeLa cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA,). Plasmid construction and transfection were performed as previously described [4]. Briefly, PLK-1 cDNA was cloned into the pcDNA3.2-DEST vector (Invitrogen), and the resulting expression plasmid (pcDNA3.2-DEST-Plk1) was verified according to the reference sequence. PLK-1 (GenBank accession no. NM_005030) siRNAs, targeting regions of the Plk-1 transcript at positions 362-384, were also used in this study. HeLa cells were transfected at 70% to 90% confluency using PLK-1 plasmid DNA (up to 4 μg) mixed with Lipofectamine 2000 (Invitrogen) at a DNA (μg)/lipid (μL) ratio of 1:2.5. Similarly, PLK-1 silencing was performed by transfecting HeLa cells with PLK-1 siRNA plasmids. At 4-6 h post-transfection, the plasmid- or siRNA-containing medium was replaced with normal culture medium containing 10% FCS, and the cells were incubated in a 5% CO2 incubator at 37°C. Transfected cells were then cultured in fresh medium for up to 12-36 h and harvested for gene expression and other assays. For cisplatin treatment, cisplatin (4 μg/ml) was added to HeLa cells, with DMSO as control. The time point chosen for the addition of cisplatin to the transfected cells was 24 h after transfection, and was based on preliminary experiments (data not shown).
Quantitative RT-PCR analysis for mRNA levels
Real-time RT-PCR was performed as detailed in our previous report [14]. Briefly, total RNA was extracted with TRIzol reagent (Invitrogen), following the manufacturer's instructions. Reverse transcription (RT) was performed, and the cDNA was synthesized from 2 μg of total RNA by using an oligo (dT)18 primer and M-MLV reverse transcriptase (TAKARA, Syuzou, Shiga, Japan) for quantitative PCR. Expression of mRNA was determined using the ABI PRISM 7300 Detection System (Applied Biosystems, Foster City, CA) and SYBR Premix Taq™ (TAKARA). The sequences of the primers were as follows: PLK1 (NM_005030) forward: 5'-GGA CTA TTC GGA CAA GTA CG-3'; PLK1 reverse: 5'-CGG AAA TAT TTA AGG AGG GTG A-3'; β-actin (NM_001101) forward: 5'-AAG ATG ACC CAG ATC ATG TTT GAG ACC-3'; β-actin reverse: 5'-AGC CAG GTC CAG ACG CAG GAT-3'. The mean value of the replicates for each sample was calculated and expressed as cycle threshold (Ct). The amount of gene expression was then calculated as the difference (ΔCt) between the Ct value of the target gene and the Ct value of β-actin.
Assessment of cell viability by MTT Assay
Treated or untreated cells were seeded into 96-well plates at 1 × 103 cells per well overnight and incubated with different concentrations of cisplatin (0 or 4 μg/ml) per treatment. After culture for 24 h, 20 μl MTT dye solution (5 mg/ml) was added to each well and samples were incubated at 37°C for 4 h. The formazan product was dissolved by adding 200 μL of DMSO to each well. The plates were read at 570 nm.
Immunoblotting analysis
Immunoblotting was performed as previously described [14]. Briefly, treated and untreated HeLa cells were collected and the protein concentrations of lysates were determined by the Bradford method (Pierce, Rockford, IL). Samples containing 10 μg of protein were boiled and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% Tris-glycine gels and transferred electrophoretically to polyvinylidene fluoride membranes. Primary antibodies (mouse anti-human PLK-1 and β-actin monoclonal antibody, 1:2,000) (Santa Cruz Biotechnology, Santa Cruz, CA) were used, followed by incubation with horseradish peroxidase-linked secondary antibody (goat anti-mouse IgG, 1:1,000). Blots were visualized using an Enhanced Chemiluminescence kit (Cell Signaling, Danvers, MA). Therelative band density of PLK-1 to β-actin was quantified with Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA). The experiment was performed in triplicate.
Cell cycle and apoptosis analysis by flow cytometry
Cell cycle and apoptosis status of HeLa cells after treatment were determined by flow cytometry. In brief, treated cells were harvested and washed once with ice-cold 0.1 M PBS, fixed with 70% ethanol and stained with PI solution (50 μg/ml propidium iodide, 1 mg/ml RNase). Cells were then analyzed for cell cycle status by flow cytometry (FACScan, Becton Dickinson, USA). To quantify apoptosis, cells were stained with annexin-V and PI using a Vybrant Apoptosis Assay Kit (Invitrogen) according to the manufacturer's instructions.
Hoechst 33258 staining and activity analysis of caspase-3
The morphological alterations associated with apoptosis were observed in transfected HeLa cells by microscopy using the Hoechst 33258 staining approach. At 36 h post-transfection, cells were fixed (methanol/glacial acetic acid at 3:1) for 15 min at 4°C. Hoechst 33258 (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well at a concentration of 10 μg/ml, and cells were then incubated for 20 min at 37°C. Before observation, cells were washed three times with PBS. Caspase-3 activation was also tested with the Caspase-3 Fluorescent Assay Kit (R&D, Minneapolis, MN). Transfected cells were harvested for the assay 36 h after transfection, according to the manual.
Statistical analyses
Immunostaining of tissue sections was analyzed with the Chi-square test. Differences between groups in terms of mRNA analysis, cell proliferation, and apoptosis were analyzed using a two-tailed t-test or analysis of variance (ANOVA) using SPSS 13.0 software. The significance level was set at P < 0.05.