Immortalized human normal cervical epithelial cells (Ect1/E6E7, CVCL_3679) and cervical cancer cell lines (SiHa, CVCL_0032; HeLa, CVCL_0030; Ca-Ski, CVCL_1100; C-33A, CVCL_1094) were purchased from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (DMEM) (Merck KGaA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (both from Beijing Solarbio Science & Technology Co., Ltd.) in a 5% CO₂ incubator at 37°C.

BiogridDataBase (version 4.4; http://thebiogrid.org/) is a database that can be used to predict the interactions between proteins. The Protein-Protein Affinity Predictor (PPA-Pred2) database (https://www.iitm.ac.in/bioinfo/PPA_Pred/prediction.html#) was used to predict the binding of PTK6 and GAB1.

C-33A cells were harvested in the logarithmic growth phase, transferred into 6-well plates, and subsequently transfected when they had reached 80% confluence. Small interfering RNA (siRNA) targeting PTK6-1 (si-PTK6-1; 5′-GCCATTAAGGTGATTTCTCGAGG-3′) and PTK6-2 (si-PTK6-2; 5′-TTCAATTGCACCTATGTTTTTAC-3′), the corresponding empty vector [siRNA-negative control (NC); 5′-AAGACAUUGUGUGUCCGCCTT-3′], the overexpression plasmid targeting GAB1 (Ov-GAB1), and its empty vector (Ov-NC) were synthesized by Geneseed Biotech Co., Ltd. According to the manufacturer's instructions, Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Shanghai, China), was used to transfect the aforementioned vectors into C-33A cells (1×10⁵ cells/well) at a concentration of 50 ng/ml at 37°C for 24 h. Following incubation, the medium was replaced with fresh DMEM and the cells were routinely cultured for 48 h. Subsequently, the cells were used to assess the transfection efficiency.

Following incubation of the transfected C-33A cells (5×10³ cells/ml) in 96-well plates for 24, 48, and 72 h, 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added and incubated with the cells for an additional 2 h. The OD values were measured using a microplate reader (BioTek^™ ELx800; BioTek Instruments, Inc.) at 450 nm and the growth curve was plotted.

The C-33A cells were seeded at a density of 1×10⁵ cells/well into 12-well plates. When the cell confluence had reached 80%, serum-free DMEM was added to the cells, which were cultured at 37°C, overnight. Subsequently, a 200-µl pipette tip was utilized to make scratches in the cell monolayer. Following 24 h of incubation, an inverted microscope (Olympus Corporation) was used to monitor the wounds. The migratory rate was calculated as the ratio of 0 h scratch width/24 h scratch width.

Matrigel (BD Biosciences) was placed in 24-well Transwell plates with 8-µm pores at 37°C for 30 min. The C-33A cells (5×10⁴ cells/ml) in 200 µl serum-starved medium were plated into the upper chamber, and the lower chamber was filled with 600 µl DMEM supplemented with 10% FBS. Following 24 h of cell culture, sterile cotton swabs were used to remove non-invading cells, while the invading cells were immobilized using 4% formaldehyde for 15 min at room temperature. The invading cells were observed in 5 random fields using an inverted microscope following staining with 0.1% crystal violet solution at room temperature for 30 min.

The induction of apoptosis was detected using a TUNEL kit (EMD Millipore). The C-33A cells were fixed using 4% paraformaldehyde at room temperature for 15 min following washing with PBS. A total of 0.3% Triton-X 100/PBS was used to permeabilize the cells for 10 min, followed by rinsing with PBS. Subsequently, 3% H₂O₂ was added to inhibit endogenous peroxidation, after which was the incubation of C-33A cells with TUNEL regent for 60 min at 37°C. 3,3-Diaminobenzidine was added to stain the cells according to the manufacturer's instructions. Finally, nuclei were stained with DAPI at room temperature for 10 min. 10 visual fields were randomly selected and an Olympus BX51 fluorescence microscope (Olympus Corporation) was used to determine the number of apoptotic cells.

C-33A cells that were in the logarithmic phase were collected and total RNA was extracted from these cells using TRIzol^® reagent (Sigma-Aldrich; Merck KGaA) according to the manufacturer's instructions. cDNA was synthesized using the QuantiTect Reverse Transcription kit (Qiagen GmbH) according to the manufacturer's instructions. An ABI 7500 Real-Time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to conduct PCR amplification according to the operating procedures of the SYBR Green PCR kit (Takara Bio, Inc.). The following thermocycling conditions were used: Initial denaturation at 95°C for 8 min; followed by 35 cycles of denaturation at 95°C for 30 sec and annealing at 60°C for 1 min; and extension for 10 min at 72°C. The nucleotide sequences used are listed as follows: PTK6 forward, 5′-TGCCCCATTGGGATGACTG-3′ and reverse, 5′-GTACAGCGCCAGGATGTGTTT-3′; GAB1 forward, 5′-GATGGTTCGTGTTACGCAGTG-3′ and reverse, 5′-CGCTGTCTGCTACCAAGTAGAA-3′; GAPDH forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′. The 2^−ΔΔCq method was used to measure the gene expression levels, which were normalized compared with those of GAPDH (20).

RIPA lysis buffer (Beyotime Institute of Biotechnology) was used to extract total protein from collected C-33A cells. The protein concentration was determined using a BCA assay and 20 µg of each protein sample was separated using 10% SDS-PAGE gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride membrane at room temperature (30-min transfer), after which 5% skim milk was used for blocking of the non-specific binding sites at room temperature for 1 h. The primary antibodies targeting PTK6 (product code ab233392; 1:1,000 dilution), Bcl-2 (product code ab32124; 1:1,000 dilution), baculoviral IAP repeat containing 5 (Birc5; product code ab76424; 1:5,000 dilution), Bax (product code ab32503; 1:1,000 dilution), matrix metalloproteinase (MMP)12 (product code ab52897; 1:1,000 dilution), MMP9 (product code ab76003; 1:1,000 dilution), GAB1 (product code ab133486; 1:1,000 dilution), and GAPDH (product code ab181602; 1:10,000 dilution) were purchased from Abcam and were incubated with the corresponding membranes at 4°C overnight. HRP-conjugated secondary antibodies (product no. 7074; 1:5,000; Cell Signaling Technology, Inc.) were used to probe the membranes at room temperature for 2 h the following day. Following the addition of the ECL solution (Vazyme Biotech Co., Ltd.; Nanjing, China), a gel imager (C150 model; Azure Biosystems, Inc.) was used to achieve visualization of the protein bands. The gray values of the protein bands were analyzed using ImageJ (v1.51; National Institutes of Health) and the relative expression levels of each protein were calculated.

For immunoprecipitation, C-33A cells were harvested and lysed in 300 µl Pierce^™ IP lysis buffer (Thermo Fisher Scientific, Inc.). A total of 400 µg protein was incubated with the antibody solution containing 2 µg PTK6 (product code ab233392; 1:30 dilution), or GAB1 (product code ab133486; 1:100 dilution), and goat anti-rabbit IgG (HRP) (product code ab205718; 1:20 dilution; all from Abcam) overnight at 4°C. Subsequently, the cell lysates were incubated for 3 h following the addition of 30 µl Protein G/A agarose beads (Invitrogen; Thermo Fisher Scientific, Inc.). Following 600 × g centrifugation at 4°C for 10 min, the pellets were washed three times with 1 ml lysis buffer before resuspension, in resuspended in 2X SDS-PAGE loading buffer. Finally, immunoblotting was used to detect immunoprecipitation products.

GraphPad Prism 7.0 software (GraphPad Software, Inc.) was used to analyze the data. The experimental data are presented as the mean ± SD. The comparison between the two groups was performed by the unpaired Student's t-test, whereas the comparison among multiple groups was performed by one-way ANOVA followed by Tukey's post hoc test. Each experiment was repeated at least three times unless otherwise specified. P<0.05 was used to indicate a statistically significant difference.

The expression levels of PTK6 were examined in human normal cervical epithelial cells (Ect1/E6E7) and in cervical cancer cell lines (SiHa, HeLa, Ca-Ski and C-33A) by RT-qPCR (Fig. 1A) and western blotting (Fig. 1B) analyses. PTK6 expression was significantly increased in cervical cancer cell lines compared with that noted in Ect1/E6E7 cells. C-33A cells were selected for subsequent experiments since they displayed the highest expression of PTK6 among SiHa, HeLa, Ca-Ski, and C-33A cells. Subsequently, PTK6 expression was knocked down and the transfection efficacy was assessed via RT-qPCR (Fig. 1C) and western blotting (Fig. 1D) analyses. siRNA-PTK6-1 was selected for subsequent experiments due to the most efficient knockdown of PTK6 expression noted in the siRNA-PTK6-1 group compared with that of the siRNA-PTK6-2 group. Following knockdown of PTK6 expression, reduced cell viability was observed compared with that of the siRNA-NC group (Fig. 1E). PTK6 depletion distinctly aggravated the induction of apoptosis in C-33A cells (Fig. 1F). In addition, western blot analysis demonstrated that the expression levels of the apoptosis-related proteins Bcl-2 and Birc5 were downregulated, while those of Bax were upregulated in C-33A cells following knockdown of PTK6 expression (Fig. 1G). These findings confirmed that the proliferation of PTK6-silenced C-33A cells was inhibited, while the induction of their apoptosis was promoted.

To confirm the effects of siRNA-PTK6-1 on the invasive and migratory activities of cervical cancer cells, wound healing and Transwell assays were performed. The cell migratory (Fig. 2A) and invasive (Fig. 2B) capacities of the siRNA-PTK6 cell group prominently decreased compared with those of the siRNA-NC group. Furthermore, the expression levels of the migration-related proteins MMP12 and MMP9 were markedly reduced in the siRNA-PTK6 group compared with those noted in the siRNA-NC group (Fig. 2C and D). The results suggested that PTK6 reduction suppressed the invasive and migratory activities of C-33A cells.

To examine further the specific mechanism of action of PTK6, its potential binding to GAB1 was predicted via the Biogrid database. The predicted binding threshold of PTK6 and GAB1 was-16.55 kcal/mol (<-5) as determined by the PPA-Pred2 database (data not shown). RT-qPCR and western blot analyses demonstrated upregulated GAB1 expression in the cervical cancer cell lines SiHa, HeLa, Ca-Ski, and C-33A, which was consistent with the expression levels of PTK6 expression in cervical cancer cells (Fig. 3A and B). Co-IP assays indicated that in the IgG group, neither PTK6 nor GAB1 proteins were precipitated, indicating that these two proteins could not bind to IgG. However, in both the PTK6 and GAB1 groups, the GAB1 or PTK6 proteins were precipitated (Fig. 3C and D). These findings indicated a strong binding affinity between the PTK6 and GAB1 proteins. Furthermore, the protein and mRNA expression levels of GAB1 in the siRNA-PTK6 group were downregulated compared with those in the siRNA-NC group, indicating a positive association between PTK6 and GAB1 (Fig. 3E and F).

To clarify whether PTK6 binds to GAB1 and to assess its involvement in the malignant progression of cervical cancer cells, an Ov-GAB1 plasmid was constructed and transfected into C-33A cells. GAB1 expression was successfully increased in the Ov-GAB1 group compared with that noted in the Ov-NC groups (Fig. 4A and B). In addition, following overexpression of GAB1, it was observed that C-33A cell viability (Fig. 4C) was increased in the siRNA-PTK6 group, while the induction of apoptosis was reduced (Fig. 4D). Moreover, western blot analysis indicated an increase in the expression levels of Bcl-2 and Birc5 and a decrease in the expression levels of Bax in C-33A cells following GAB1 overexpression (compared with siRNA-PTK6 + Ov-NC; Fig. 4E).

Overexpression of GAB1 caused a partial reversal of the suppressive effects of PTK6 knockdown on the migration and invasion of C-33A cells (Fig. 5A and B). Moreover, the expression levels of MMP12 and MMP9 were increased in C-33A cells co-transfected with Ov-GAB1 and siRNA-PTK6 compared with those noted in the siRNA-PTK6 + Ov-NC group (Fig. 5C and D). In general, the overexpression of GAB1 could counteract the reduced migratory and invasive activities observed following knockdown of PTK6 expression in C-33A cells.