SiHa and SiHa-DPP cells were purchased from Professor Wang He (West China Second University Hospital, Sichuan, China). The SiHa cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China) and 50 U/ml penicillin and streptomycin (all from Beyotime Institute of Biotechnology, Haimen, China). Human normal oral keratinocytes were cultured in Oral Keratinocyte Medium (ScienCell Research Laboratories, Carlsbad, CA, USA) containing 5 ml oral keratinocyte growth supplement and 5 ml penicillin/streptomycin solution. Transfection was performed when cells had reached ~80% confluency. P16 shRNA (shP16) and the control shRNA (0.1 mg; mock) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) vectors were transfected with lipofectamine LTX reagent with PLUS reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Following transfection, the cells were isolated using culture medium. Western blot analysis was then performed to determine the efficiency of p16 knockdown.

Total RNA was isolated using TRIzol Reagent (Invitrogen Life Technologies) and reverse transcription was performed using the PrimeScript™ First Strand cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s instructions. cDNA was generated from 5 mg of total RNA, also according to the manufacturer’s instructions. Reverse transcription (RT) real-time quantitative polymerase chain reaction (qPCR) was performed to evaluate the expression levels of P16 mRNA in the SiHa cell lines. Quantitative PCR was performed using Takara SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) and an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The total reaction volume was 10 μl consisting of 5 μl 2× SYBR premix EX Taq™, 0.5 μl (10 μM) PCR forward primer, 0.5 μl (10 μM) PCR reverse primer, 0.5 μl cDNA and 3.5 μl dH₂O. The sequences of the gene specific primers were as follows: Forward, 5′-CCTTTGGTTATCGCAAGCTG-3′; and reverse, 5′-CCCTGTAGGACCTTCGGTGA-3′ for P16; forward, 5′-CAAGGGTCATTATGGGTTAGGC-3′ and reverse, 5′-TTAGGTGTAGGGGAGGGGAGA-3′ for pRb; and forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′ for GAPDH.

Proteins were extracted using cell lysis buffer (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. The protein concentration was quantified using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). For western blot analysis, equal amounts of total protein (20 μg) were boiled and separated by SDS-PAGE. Following electrophoresis, the protein was blotted onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) and blocked for 2 h at room temperature. The membranes were then incubated with human anti-mouse monoclonal p16 antibody (Sigma-Aldrich, St. Louis, MO, USA), human anti-mouse polyclonal pRb and β-actin antibodies (Cell Signaling Technology Inc., Danvers, MA, USA), human anti-rabbit monoclonal CDK4 antibody (Cell Signaling Technology, Inc.) overnight at 4°C. The membranes were then washed with Tris-buffered saline three times prior to incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit CDK4 and HRP-conjugated goat anti-mouse p16, Rb and β-actin secondary antibodies (Beyotime Institute of Biotechnology) and visualized using an ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were scanned using a myECL Imager (Thermo Fisher Scientific) and the relative level of protein expression was analyzed by ImageJ software (imagej.nih.gov/ij/).

Cells proliferation was measured by MTT assay (Funakoshi Co., Tokyo, Japan) (20) in 96-well microculture plates (Thermo Fisher Scientific). The cells were seeded at a density of 1×10⁴ cells/well in 96-well plates in DMEM containing 10% FBS. Three duplicate wells were set up for each group and the experiment was peformed in triplicate. After 48 h incubation, 20 μl MTT solution (5 mg/ml) in phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology), was added to each well for 4 h. The absorbance of each well was analyzed using an Infinite F50 microplate reader (Tecan, Männedorf, Switzerland) at a wavelength of 570 nm. Proliferation curves were plotted according to the optical density.

Apoptosis detection was carried out using Annexin V flow cytometry (Becton Dickinson, San Jose, CA, USA) as previously described (21). Cells were treated with 2 mM DDP or left untreated, and adherent cells were then harvested after 72 h and washed in cold PBS, centrifuged at 200 × g at 4°C for 5 min and resuspended in Annexin-binding buffer. Annexin V and propidium iodide (Invitrogen Life Technologies) were added to the cell resuspension and the stained cells were analyzed by flow cytometry using the S3 cell sorter (Bio-Rad, Hercules, CA, USA) at wavelengths of 530 and >575 nm.

Statistical significance was determined using SPSS, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Data are expressed as the mean ± standard error of the mean.

The DDP-sensitive and -resistant cervical carcinoma cell lines were used to compare DDP responses. MTT assay and flow cytometry were performed to identify the characteristics of human cervical cancer cells (SiHa) and SiHa-DDP cells. As shown in Fig. 1A, reduced cellular growth was observed in the SiHa-DDP cells when compared with SiHa cells, and less apoptosis was observed in SiHa-DDP cells following treatment with DDP (Fig. 1B). qPCR demonstrated that the levels of P16^INK4A increased (Fig. 1C) while cyclin D1 and pRb levels were downregulated in SiHa-DDP cells when compared with SiHa cells. These results indicate that SiHa cells resistance to DDP was associated with high P16^INK4A expression levels.

It has been hypothesized that the cyclin D1-CDK4-pRb pathway causes DDP resistance via cell cycle arrest (14,15). Therefore, the influence of P16 on the cyclin D1-CDK4-pRb pathway in SiHa-DPP cells was analyzed. Co-immunoprecipitation was performed to evaluate the interaction between P16^INK4A and CDK4 in SiHa-DDP cells and SiHa cells. The results showed that P16 was coprecipitated with CDK4 in both cell lines (Fig. 2A), and the interaction in SiHa-DDP was enhanced when compared with SiHa cells, indicating that the P16-CDK4 complex existed and may be involved in DDP-resistance. To investigate whether the enhanced levels of the P16-CDK4 complex contributes to cell cycle arrest, western blot analysis was performed to detect changes in pRb activity. As shown in Fig. 2B, pRb levels decreased concurrently with the upregulation of P16^INK4A protein levels in SiHa-DDP cells, indicating that P16 may compete with cyclin D1 to interact with CDK4, thus inhibiting pRb activation associated with DDP-resistance in SiHa-DDP cells.

To investigate whether P16^INK4A regulates cellular growth and apoptosis in SiHa-DPP cells and contributes to DDP-resistance, shRNA and mock of P16^INK4A were transfected into SiHa-DPP cells. Western blot analysis was used to evaluate the protein levels of P16^INK4A. It was indicated that P16^INK4A in shRNA-transfected cells was markedly downregulated when compared with mock-transfected cells (Fig. 3A). To investigate the proliferative effects in shP16-transfected cells, cellular growth was monitored for 144 h. The shP16-transfected SiHa-DPP cells exhibited a significant increase in cellular growth when compared with mock-transfected cells (P=0.05; Fig. 3B). Therefore, the effect of DDP treatment on the sensitivity of P16^INK4A knockdown cells to DNA damage was assessed. Flow cytometry revealed that shP16-transfected cells were more sensitive to DDP-induced apoptosis when compared with mock-transfected cells (Fig. 3C).

To identify the mechanism by which downregulated P16^INK4A influences proliferation, which contributes to DDP resistance, the protein expression of pRb and cyclin D1 in SiHa, mock-transfected SiHa-DDP cells and shP16-transfected SiHa-DDP cells was investigated. pRb and cyclin D1 were significantly increased in shP16-transfected SiHa-DDP cells (Fig. 4).