The human embryonic kidney cell line 293 and the human cervical cancer cell lines SiHa, CaSki, and HeLa were obtained from the American Type Culture Collection. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS; 10%; both obtained from Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 μg/ml streptomycin (both obtained from HyClone; GE Healthcare) at 37°C in a humidified atmosphere of 5% CO₂. 293 cells were used for adenovirus amplifica tion.

Adenovirus expressing S100A9 and green fluorescent protein (AdS100A9), adenovirus expressing small interfering (si)RNA targeting β-catenin and red fluorescent protein (Adsiβ-catenin), control adenovirus expressing green fluorescent protein (AdGFP) and red fluorescent protein (AdRFP), and pGST-moluc and pGST-moluc-hS100A9 plasmids were kindly donated by Professor T.C. He (The University of Chicago Medical Center, Chicago, IL, USA). Competent bacteria E. coli (BL21) were saved in our laboratory. Adenoviruses expressing siRNA targeting S100A9 and red fluorescent protein (AdsiS100A9), and control adenoviruses expressing red fluorescent protein (AdsiControl) were constructed in house. The kit used for semi-quantitative PCR was purchased from Takara Bio, Inc. Antibodies, including mouse anti-β-actin, anti-vimentin and anti-β-catenin were purchased from Santa Cruz Biotechnology, Inc. (cat. nos. sc-47778, sc-66001 and sc-59737). Rabbit anti-S100A9 antibody was purchased from Abcam (cat. no. ab92507). Rabbit anti-E-cadherin antibody was purchased from ImmunoWay (cat. no. YM3353, Plano). Rabbit anti-histone H3 antibody was purchased from Abmart (cat. no. P30266). Secondary antibody reagents, such as goat anti-mouse IgG serum and goat anti-rabbit IgG serum were obtained from Beijing Zhongshan Golden Bridge Biotechnology (cat. no. 2305 and no. 2301). Western blot reagents and radioimmunoprecipitation assay (RIPA) buffer were purchased from Beyotime Institute of Biotechnology. Phosphatase and protease inhibitors were purchased from Roche Diagnostics GmbH. Polyvinylidene difluoride (PVDF) membranes and an enhanced chemiluminescence (ECL) kit were purchased from EMD Millipore.

HeLa cells were infected with AdS100A9 and AdGFP, whereas SiHa cells were infected with AdsiS100A9 and AdsiControl. After 8-12 h of incubation, the medium was replaced with complete medium containing FBS followed by continued cell culture for subsequent experiments. The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

The pGST-moluc and pGST-moluc-hS100A9 plasmids used in the present study has been described previously (4). In brief, pGST-moluc and pGST-moluc-hS100A9 was transfected into E. coli (BL21) by calcium chloride-mediated transformation. Isopropylthio-β-D-galactoside was used to induce the expression of GST and GST-hS100A9 proteins. The bacteria were then collected and sonicated on ice at 4°C. The supernatants were incubated with glutathione-sepharose 4B beads, GST and GST-hS100A9 proteins on the beads were eluted by elution buffer with reduced glutathione on ice. Finally the GST and GST-hS100A9 proteins were filtered and stored at −80°C. Cells were treated with 20 μg/ml of recombinant proteins for 48 h at 37°C.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA samples were synthesized using random primers and a Reverse Transcriptase PCR kit (Takara Bio, Inc.) according to the manufacturer's protocols. cDNA was used as a template for semi-quantitative PCR. The thermocycling conditions were as follows: 94°C for 5 min, 94°C for 30 sec, 68°C for 30 sec and 72°C for 12 cycles with a decrease in 1°C/cycle; then, 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec for 18-27 cycles depending on the abundance of the target genes. The PCR products were identified by electrophoresis using 1.5% agarose gels. GAPDH was used as an internal reference control. The results were recorded using Gel Doc 1000 imaging system and Quantity One version 4.5.0 software (Bio-Rad Laboratories, Inc.). The primers used in this study are shown in Table I.

Total cellular protein of cervical cancer cells were prepared using RIPA buffer containing phosphatase and protease inhibitors. Nuclear and cytoplasmic proteins were extracted using Nuclear-Cytosol Extraction Kit (cat. no. KGP1100, Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Protein extracts were separated by 6-15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin at 37°C for 2 h, washed with Tris-buffered saline with Tween-20 (TBST) and incubated with primary antibodies against S100A9, vimentin, E-cadherin, β-catenin, histone H3, and β-actin separately (1:1,000 dilution) at 4°C overnight. After washing with TBST, the membranes were incubated with the a secondary antibody conjugated with horseradish peroxidase (1:5,000 dilution) for 1 h at 37°C. Finally, the blots were washed with TBST, and then visualized using the Bio-Rad Gel Doc 1000 imaging system and analyzed by Quantity One Version 4.5.0.

Cell proliferation was analyzed via an MTT assay (Promega Corporation). A total of 3.5×10³ HeLa cells or 4.5×10³ SiHa cells were seeded in 96-well flat-bottomed microplates. After infection with or without adenoviruses in DMEM containing 1% FBS for 24, 48 and 72 h, 10 μl of MTT reagent was added to each well and incubated at 37°C for 4 h. Dimethyl sulfoxide (100 μl) was added to dissolve the formazan crystals for 10 min at room temperature. Finally, the absorbance was measured at 490 nm using an automatic enzyme immunoassay analyzer (Bio-Rad Laboratories, Inc.). The experiments were performed five times in three independent experimental trials.

Cervical cancer cells were seeded in 6-well plates and incubated at 37°C until the cells attained 90% confluence. The cells were then infected with adenoviruses. Cell monolayers were carefully wounded by scratching with a sterile 10-μl pipette tip. The cells were then washed twice with cold PBS and incubated in serum-free DMEM at 37°C. Wound areas were observed and imaged at ×100 magnification at 0, 24 and 36 h with an inverted fluorescent microscope (Nikon eclipse 80i; Nikon Corporation). Overall, the experiments were repeated three times.

A chamber of non-type I-collagen- coated 24-well Transwell cell culture inserts (EMD Millipore) was used for the Transwell migration assay. Briefly, after infection with or without adenoviruses and recombination proteins for various durations, the cells were trypsinized, washed, and suspended in 400 μl of serum-free DMEM and finally seeded in the upper chamber (HeLa, 4×10⁴ cells per well; SiHa, 6×10⁴ cells per well). The lower chamber was coated with 700 μl of DMEM containing 20% FBS as a chemoattractant. After 24 h, cells were fixed with methanol for 20 min and stained with 0.05% crystal violet for 30 min at room temperature. Cells on the upper surface of the insert membrane were removed with cotton swabs. Finally, the cells were counted using an inverted microscope (magnification, ×100) in five randomly selected fields for each well. The experiment was repeated three times.

The cells were seeded on sterile glass coverslips in 24-well culture plates. After treatment with or without AdS100A9, cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton X-100 at 37°C for 30 min. After blocking with goat serum at 37°C for 30 min, the cells were incu- bated with antibodies against E-cadherin, N-cadherin (1:100 dilution in PBS) at 4°C overnight. Subsequently, slides were washed with PBS and incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h at room temperature in the dark. Then, the cells were washed with PBS, counter stained with DAPI (Beyotime Institute of Biotechnology) for 5 min at room temperature, washed with PBS again and then the coverslips were mounted using antifade mounting medium. The fluorescence of the various groups was visualized imaged with a Nikon eclipse 80i inverted fluorescent microscope (Nikon Corporation).

All data are presented as the mean ± standard deviation. Differences were analyzed using one-way ANOVA followed by a Tukey's multiple comparison test. All statistical analyses were performed using SPSS statistical package 17.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software, Inc.). P≤0.05 was considered to indicate a statistically significant difference.

We first evaluated the endogenous expression of S100A9 in SiHa, CaSki, and HeLa cell lines using semi-quantitative PCR and western blot analysis. As shown in Fig. 1A, the mRNA expression levels of S100A9 in HeLa cells were significantly reduced than in SiHa and CaSki cells (P≤0.001 and P≤0.01). These results were confirmed by western blot analysis (P≤0.01; Fig. 1B). Subsequently, semi-quantitative PCR and western blot analysis (Fig. 1C and D) were performed to detect the expression of S100A9 in HeLa and SiHa cells infected with AdS100A9 and AdsiS100A9 for 48 h, respectively. The expression of S100A9 observed in the AdS100A9 group was significantly increased compared with the AdGFP and Blank groups in HeLa cells (P≤0.001 and P≤0.05), whereas a significant decrease in S100A9 expression was detected in the AdsiS100A9 group compared with AdsiControl and Blank groups in SiHa cells (P≤0.05 and P≤0.01). These results indicated that S100A9 was successfully overexpressed in HeLa cells and downregulated in SiHa cells via infection with AdS100A9 and AdsiS100A9, respectively.

To investigate the effects of S100A9 on the proliferation of cervical cancer cells, we treated the cells with AdS100A9 and AdsiS100A9 for 3 days and examined by an MTT assay (Fig. 2). Compared with the control groups, the proliferation of HeLa cells was significantly enhanced in the S100A9-overexpressing group on day 3 (P≤0.05; Fig. 2A). On the contrary, the proliferation of SiHa cells was significantly inhibited in the S100A9-knockdown group compared with the control (P≤0.05; Fig. 2B). These results demonstrated that overexpressed S100A9 could promote the proliferative potential of cervical cancer cells.

We further studied the role of S100A9 in the migration of cervical cancer cells by wound healing and Transwell migration assays. The results revealed that the wound closure rate of S100A9-overexpressing HeLa cells was notably increased than the control group, whereas that of SiHa cells was impaired following knockdown of S100A9 (Fig. 3A). Similar results were observed in the Transwell migration assay. The number of HeLa cells migrated across the membrane in AdS100A9 group was significantly higher than that in the AdGFP group (P≤0.05; Fig. 3B). These results are consistent with the wound healing ability of SiHa cells. The number of S100A9-depleted SiHa cells that migrated across the membrane was significantly lower compared with the control (P≤0.01). Collectively, these results suggested that upregulated S100A9 expression may facilitate the migration of cervical cancer cells.

Emerging evidences support that EMT has a crucial role in the metastasis of primary tumors. To evaluate whether S100A9 promoted EMT in cervical cancer cells, western blot and immunofluorescence analyses were conducted to detect the levels of E-cadherin and vimentin. As presented in Fig. 4A, overexpression of S100A9 in HeLa cells significantly decreased the expression of E-cadherin (P≤0.05), and increased that of of vimentin (P≤0.01). On the contrary, knockdown of S100A9 in SiHa cells significantly decreased the expression of vimentin (P≤0.01), and increased that of E-cadherin (P≤0.05). These results were further confirmed by immunofluorescence assays (Fig. 4B). These findings suggested that S100A9 is involved in the regulation of EMT-related protein expression, and may act as a predictive factor and therapeutic target for preventing metastasis in cervical cancer.

It is well known that aberrant activation of the Wnt/β-catenin pathway serves a critical role in the development of cervical cancer. To investigate whether S100A9 was involved in the regulation of Wnt/β-catenin pathway in cervical cancer cells, western blot and semi-quantitative PCR analyses were performed to study the effects of S100A9 on the expression of β-catenin protein, and the transcriptional levels of c-Myc, Snail, and twist-related protein-1 (Twist), which are the classical target genes of Wnt/β-catenin pathway. As shown in Fig. 5A, after infecting HeLa cells with AdS100A9, overexpression of S100A9 significantly enhanced the accumulation of total β-catenin and nuclear β-catenin (P≤0.01), but knockdown of S100A9 via the infection of SiHa cells with AdsiS100A9 decreased the accumulation of total β-catenin and nuclear β-catenin compared with the control groups (P≤0.001 and P≤0.05). Additionally, overexpression of S100A9 caused by infecting HeLa cells with AdS100A9 significantly promoted the transcription of c-Myc (P≤0.05), Snail (P≤0.01), and Twist (P≤0.05) compared with the control. Conversely, the mRNA levels of the aforementioned target genes of the Wnt/β-catenin signaling pathway were significantly reduced when SiHa cells were infected with AdsiS100A9 compared with the control (P≤0.01, P≤0.01 and P≤0.001, respectively; Fig. 5B). These results demonstrated that S100A9-induced upregulation of c-Myc, Snail, and Twist expression in cervical cancer cell lines may be associated with the accumulation of total β-catenin and nuclear β-catenin.

To determine whether Wnt/β-catenin pathway is involved in S100A9-induced promotion of proliferation, migration and EMT in cervical cancer cells, adenovirus Adsiβ-catenin was used as the antagonist to impair the activation of the Wnt/β-catenin pathway. As presented in Fig. 6A, the effects of S100A9 on the proliferation of HeLa cells was investigated by an MTT assay. After the treatment of cells with GST-hS100A9 at 10 and 20 μg/ml for 72 h, the proliferation of HeLa cells was significantly increased than that of the control groups (P≤0.05). Additionally, the results further showed a more notable effect in response to high concentration recombinant protein; thus, we selected 20 μg/ml of recombinant protein for the following experiments. As shown in Fig. 6B, β-catenin expression was significantly downregulated by adenovirus Adsiβ-catenin in HeLa cells compared with the control (P≤0.05). After treatment with GST-hS100A9 and adenovirus Adsiβ-catenin, the mRNA levels of Snail, and Twist were significantly reduced in HeLa cells compared with the control groups (P≤0.05 and P≤0.01; Fig. 6C). In addition, treatment with Adsiβ-catenin significantly inhibited the proliferation and migration of HeLa cells induced by GST-hS100A9 (P≤0.05 and P≤0.01; Fig. 6D and E). Furthermore, the expression of EMT markers was examined in HeLa cells after treatment with and without AdRFP, Adsiβ-catenin, GST and GST-hS100A9. We observed that downregulation of β-catenin significantly suppressed the effects of S100A9 on the expression of E-cadherin and vimentin compared with the control groups (P≤0.05 and P≤0.01; Fig. 6F). These findings demonstrated that S100A9 induced promotion of proliferation, migration and EMT through activation of Wnt/β-catenin pathway in cervical cancer cells.