Residues of 149 ThinPrep-processed cervical cytology samples were collected from the International Peace Maternity and Child Hospital, Affiliated to Shanghai Jiao Tong University (Shanghai, China), from February 2010 to February 2011. The age of the patients was between 22 and 60 years (mean age, 35 years); none of the patients were pregnant, and none had a history of cervical surgery or pelvic radiation therapy. The samples included 20 cases of high-grade squamous intraepithelial lesions (HSILs), 42 cases of low-grade squamous intraepithelial lesions (LSILs), 50 cases of atypical squamous cells of undetermined significance (ASCUS), 17 cases of negative samples and 20 cases of invasive squamous cell carcinoma (ISCC). The diagnosis and histological classification of the cervical cancer samples were carried out according to the criteria proposed by the International Federation of Gynecology and Obstetrics (FIGO). The patients, including 35/50 (70%) ASCUS cases and 36/42 (86%) of the LSIL cases were subjected to cytological and histological analyses within 2 years of the initial diagnosis. The use of cytological residues was approved by the Human Investigation Ethical Committee of the International Peace Maternity and Child Health Hospital Affiliated to Shanghai Jiao Tong University. The residues of the cervical cytological samples were centrifuged at 1,500 rpm for 3 min, followed by incubation with agarose solution (agarose:H₂O, 1 g:50 ml). Paraffin sections (5-mm³-thick) were obtained after cooling down the samples.

Thin-layer cytology slides were dried overnight in an oven at 37°C. Antigen retrieval was performed by heating the immersed slides in 10 mM sodium citrate buffer, pH 6.0, at 951°C in a microwave oven for 15 min. Endogenous peroxidase was blocked by treatment with 3% H₂O₂ in methanol. Rabbit monoclonal antihuman p16^ink4a (Epitomics, Burlingame, CA, USA) antibodies were used at a dilution of 1:1,000. Antibody binding was detected using EnVision reagents (Boster Bio-Engineering Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. A light hematoxylin counterstain was used. The primary antibody was replaced with preimmune IgG serum and used as a negative control. HeLa cells were used as the positive control. The experimental conditions were uniform for all the samples. Morphological criteria based on a modified scoring system described in the study by Wentzensen et al (16) were used for the assessment of the immunoreactivity of the cells for p16^ink4a. More than 10 epithelial groups were assessed in each slide with the highest immunoreactivity. Cells with atypical cytological features were evaluated for immunoreactivity and an index of 0–4 was obtained. The immunoreactivity for p16^ink4a in the samples was evaluated by 2 of the authors. Cases with discrepancy in scoring were resolved under a multihead microscope (Zeiss Axioskop 40; Zeiss, Oberkochen, Germany). The specific cytological diagnosis of the samples was disclosed after the scoring.

Human cervical cancer cell lines (SiHa and Hela) were obtained from the Committee on Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (11030; Gibco, Auckland, New Zeland) supplemented with 10% fetal bovine serum (FBS) (16000–44; Gibco, Carlsbad, CA, USA).

In order to inhibit the expression of endogenous p16^ink4a, we designed and prepared an HPLC-purified p16^ink4a-siRNA, based on the p16^ink4a gene sequence. A scrambled siRNA with no homology to any known human mRNA was used as a negative control. siRNA oligonucleotide duplexes were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of the p16^ink4a-siRNA oligonucleotides were as follows: forward, 5′-CGCACCGAAUAGUUACGG UTT-3′ and reverse, 5′-ACCGUAACUAUUCGGUGCGTT-3′. The cells were seeded in 12-well plates till they reached 70–80% confluence and wer grown overnight prior to transfection. The transfection of the SiHa and Hela cells with the p16^ink4a-siRNA or the non-target control-siRNA was accomplished using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The experimental groups were classified into the p16^ink4a-siRNA, the control-siRNA and the untreated group.

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies; Shanghai, China) and reverse transcribed using a reverse transcriptase kit (Takara Biotechnology, Dalian, China). Gene expression detection was performed using SYBR-Green Master mix (Takara) on using an ABI Prism 700 thermal cycler (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Gene expression was calculated using the 2^−ΔΔCt formula. The primer sequences used were as follows: p16^ink4a forward, 5′-CTTCCTGGACACGCTGGT-3′ and reverse, 5′-ATCTATGCGGGCATGGTTACT-3′; and β-actin forward, 5′-CAGCCATGTACGTTGCTATCCAGG-3′ and reverse, 5′-AGGTCCAGACGCAGGATGGCATG-3′. Duplicate reactions were performed for each sample, and the same experiment was repeated 3 times, with β-actin as a reference gene.

Cells were grown in 10-cm dishes. After 2 rinses in ice-cold PBS, the cells were physically harvested and lysed in ice-cold HNTG buffer [50 mmol/l HEPES (pH 7.5), 150 mmol/l NaCl, 10% glycerol, 1% Triton X-100, 1.5 mmol/l MgCl₂, 1 mmol/l EDTA, 10 mmol/l sodium PPI, 100 μmol/l sodium orthovanadate, 100 mmol/l NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin and 1 mmol/l PMSF] on ice for 30 min. The total protein content was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Protein samples (20 μg) were subsequently separated on 12% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels and electrotransferred onto PVDF membranes. The membranes were blocked and then probed with antibodies against p16^ink4a (1:500 dilution), Rb (1:1,000 dilution) (both from Epitomics), phosphorylated Rb (phopsho-Rb; 1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (1:10,000 dilution; Epitomics), caspase-3 (1:6,000 dilution; Cell Signaling Technology). Data were normalized to β-actin expression (1:1,000; Proteintech Group, Inc., Chicago, IL, USA) by densitometry. Statistical data from at least 3 experiments were analyzed for additional validation.

The cells were seeded into 96-well plates at 2×10⁵ cells/ml and cultured in DMEM/Ham’s F12 medium supplemented with 10% FBS for 1–5 days. Cell growth was documented every 24 h via a colorimetric assay using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, MO, USA). The absorbance was determined at 490 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The control samples were treated with the vehicle (0.1% DMSO or ethanol in DMEM/Ham’s F12 culture medium). In each individual experiment, proliferation was determined in triplicate, and the overall experiment was repeated at least 3 times.

For wound healing experiments, cells (5×10⁶/well) were seeded in 6-well plates and allowed to adhere for 24 h. Cell monolayers were scarred with a sterile micropipette tip and incubated in serum-free medium for 24 h. Three defined areas of each sample were monitored, and photographed at 0, 6, 12 and 24 h. The migration index was calculated from the distance traveled by the cell monolayer relative to the gap created with the pipette tip at 0 h, for the untreated, control-siRNA- or the p16^ink4a-siRNA-treated cells, as previously described (17).

Experiments were performed using 6.5-mm Transwell chambers equipped with 8.0 μm pore-size polycarbonate membranes (Corning, Inc., Glendale, AZ, USA). Cells (1×10⁵) were added to the top section of a Boyden chamber apparatus in 150 ml serum-free DMEM medium. Subsequently, 600 ml complete medium were added to the lower chamber. Following 10 h of incubation, the cells that had attached to the underside of the membrane were fixed with 4% paraformaldehyde. The cells were stained with 5% crystal violet and counted at 200-fold magnification. The migration assay was repeated at least 3 times.

The upper chambers were first coated with 40 ml of Matrigel at a 1:3 dilution (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37°C for 2 h, followed by the addition of 1×10⁵ cells to the top section of a Boyden chamber apparatus in 150 ml serum-free DMEM medium and 600 ml complete medium to the lower chamber. Folowing 24 h of incubation, the cells that had attached to the underside of the membrane were fixed with 4% paraformaldehyde. The cells were stained with 5% crystal violet and counted at 200-fold magnification. The invasion assay was repeated at least 3 times.

Approximately 1×10³ cells were seeded into each well of a 6-well culture plate and incubated for 14 days at 37°C, followed by washing in PBS twice and staining with crystal violet. The number of colonies containing ≥50 cells was counted under a light microscope, and calculated as follows: Plate clone formation efficiency (%) = (number of colonies/number of cells inoculated) ×100. Each experiment was performed in triplicate.

The cells were harvested and subjected to dual staining with Annexin V and propidium iodide (PI) using an Annexin V-PI Apoptosis Detection kit (KeyGEN Biotech Co., Ltd., Nanjing, China). Separately, the cells were harvested and subjected to staining with PI using a Cell Cycle Detection kit (KeyGEN Biotech Co.). The cells were treated according to the manufacturer’s instructions. The resulting fluorescence intensities were measured by flow cytometry using a Beckman Coulter FC 500 flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Experiments were performed in triplicate and reproducibility was confirmed in 3 independent experiments.

All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 17.0 (SPSS, Inc., Chicago, IL, USA). The significance of the differences in p16^ink4a expression among all cervical cytology diagnostic categories was examined using a non-parametric Kruskal-Wallis H test and an χ² test. The significance of the differences in p16^ink4a expression between any 2 diagnostic groups was analyzed by the Student-Newman-Keuls (SNK) test. Data are presented as the means ± standard deviations (SD). Data were assessed using a Student’s t-test or one-way ANOVA analysis for multiple comparisons. Values of P<0.05 or P<0.01 or P<0.001 were considered to indicate statistically significant differences. All experiments were carried out in triplicate and repeated at least 3 times.

The immunoreactivity of p16^ink4a was predominantly localized in the nuclei, although a weak cytoplasmic expression was observed focally and occasionally (Fig. 1A). The normal squamous cells were negative for p16^ink4a. The focal weak to moderate immunoreactivity of p16^ink4a was noted in the ASCUS and LSIL samples. The HSIL and ISCC samples were often strongly positive for p16^ink4a. A comparison of the p16^ink4a index in the different categories of cervical cytology diagnosis is summarized in Fig. 1B. A statistically significant difference was observed in the p16^ink4a index among the different diagnostic categories (P<0.001, Kruskal-Wallis test). The ASCUS and LSIL samples showed a significantly higher p16^ink4a index (P<0.05, SNK test) compared with the negative cervical cells, although no significant difference in the p16^ink4a index was noted between the ASCUS and LSIL categories. No significant difference was observed in the p16^ink4a index between the HSIL and ISCC samples. However, the HSIL samples showed a significantly higher p16^ink4a index than the ASCUS and LSIL samples (P<0.05, SNK test).

To determine whether p16^ink4a enables the evaluation of the likelihood of progression in a particular CIN, we compared the p16^ink4a expression of the ASCUS and LSIL cases manifesting different pathologies. The cytology and histologyical follow-up data of the patients within 2 years after the initial diagnosis were available for 35/50 (70%) of the ASCUS and 36/42 (86%) of the LSIL cases (Table I). Among the ASCUS cases, the expression of p16^ink4a (P<0.05, SNK test) in cases diagnosed with CIN1 and CIN2–3 was significantly higher compared with the benign lesions. Among the LSIL cases, p16^ink4a (P<0.05, SNK test) expression in cases diagnosed with CIN1 and CIN2–3 was also significantly higher compared with the benign lesions. On the other hand, no significant difference was observed in the presence of high-risk (HR)-HPV (detected by the Digene test) among cases with a pathological diagnosis of CIN1, CIN2–3 and benign lesions. These results indicate a strong association between p16^ink4a overexpression and the development of cervical precancerous lesions. p16^ink4a may be particularly useful in the triage of patients with borderline smears reported as ASCUS and LSIL, suggesting that a high p16^ink4a immunoreactivity correlates with a greater likelihood of progression to CIN1 and CIN2–3 lesions in the future, independent of their HPV status.

To further explore the role of p16^ink4a in cell proliferation and apoptosis, the cervical cancer cell lines (SiHa and HeLa) were transfected with p16^ink4a-siRNA. As shown in Fig. 2A and B, the mRNA and protein expression of p16^ink4a was decreased significantly (P<0.05). The effects of the p16^ink4a downregulation on cell proliferation were detected by MTT assay (Fig. 2C). MTT assay revealed that the downregulation of p16^ink4a markedly decreased SiHa and HeLa cell growth. We then investigated whether p16^ink4a is required in anchorage-dependent growth. In the SiHa and HeLa cells transfected with the p16^ink4a-siRNA, plate colony formation assay showed a significant decrease in the number and size of colonies compared with the cells transfected with the control-siRNA or the untreated cells (Fig. 2D and E). These findings indicate that the downregulation of p16^ink4a inhibits cervical cancer cell proliferation.

In order to further determine the role of the downregulation of p16^ink4a in cell migration, wound healing assays were performed (Fig. 3A–D). In the untreated SiHa and HeLa cells or those treated with the control-siRNA, the wound had almost closed after 12 h of incubation. However, the SiHa (P<0.001) and HeLa (P<0.05) cells treated with p16^ink4a-siRNA were unable to do so after 12 h of incubation. Furthermore, the results of migration and invasion assay measured in the Transwell chambers were consistent with the wound healing assay. After 10 or 24 h of incubation, migration and invasion were markedly decreased in the SiHa and HeLa cells treated with p16^ink4a-siRNA (Fig. 3E–H). These results suggest that the downregulation of p16^ink4a affects tumor metastasis in SiHa and HeLa cells.

The SiHa cells were analyzed by Annexin V-PI staining and flow cytometry 48 h following treatment with p16^ink4a-siRNA. A greater percentage of the SiHa cells transfected with p16^ink4a-siRNA were apoptotic, compared with those treated with the control-siRNA or the untreated group (Fig. 4A and C). The cell cycle was also determined by flow cytometry after PI staining. The results revealed that a greater number of SiHa cells transfected with p16^ink4a-siRNA for 48 h remained in the G1 phase, compared with those treated with the control-siRNA or the untreated group (Fig. 4B and D). These data suggest that the downregulation of p16^ink4a promotes apoptosis and G1 cell cycle arrest in cervical cancer cells. To further elucidate the mechanisms of action of p16^ink4a, we examined the protein expression of the p16^ink4a-cyclin-Rb pathway in the SiHa cells transfected with p16^ink4a-siRNA. Western blot analysis was used to detect changes in the expression of cell cycle regulatory proteins (Fig. 4E). We found that the downregulation of p16^ink4a in the SiHa cells markedly induced the activation of caspase-3, a crucial mediator of programmed cell death. The expression of cyclin D1 was also decreased; however, the levels of total Rb (tRb) and phospho-Rb protein were not significantly altered. These data suggest that the anti-tumor effects of the p16^ink4a downregulation are caspase-3- and cyclin D1-dependent.