The interaction between NUAK2 and CYFIP2 was predicted using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (version 11.0, http://string-db.org/) (13). The UALCAN cancer database (http://ualcan.path.uab.edu) was used to retrieve data regarding the expression levels of NUAK2 and CYFIP2 in the primary tumor tissues (n=305) of patients with cervical squamous cell carcinoma (CESC) and normal tissues (n=3) from healthy controls (14); these databases were from The Cancer Genome Atlas.

CC tissues and pair-matched non-cancerous cervical tissues (2 cm from the lesions) were obtained from 36 patients (age, 30–65 years) diagnosed with CC at Wuhan Children's Hospital (Wuhan, China) from April 2018 to September 2019. Patients were excluded from the study if they received chemotherapy or radiotherapy prior to surgery. Written informed consent was obtained from all patients.

The HeLa cervical adenocarcinoma cell line, C-33A, SiHa, CaSKi and HCC-94 cervical squamous cell carcinoma cell lines, and Ect1 human normal cervical cell line were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, 100 µg/ml penicillin and 100 µg/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C in an incubator containing 5% CO₂. The medium was changed every 2 days and cells were grown to logarithmic phase for use in subsequent experiments.

Two small interfering RNA (siRNA) sequences against CYFIP2 (si-CYFIP2-1/si-CYFIP2-2) and a nontargeting siRNA used as a negative control (si-NC) were designed by Shanghai GenePharma Co., Ltd. Targeting and nontargeting sequences were as follows: si-CYFIP2-1, 5′-AGGCTAACTTTGACACAAACT-3′; si-CYFIP2-2, 5′-GGCTAACTTTGACACAAACTT-3′; si-NC, 5′-GCGTTCTTAACTTTGAACC-3′. siRNAs were transfected into cells using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, HeLa cells were seeded at a density of 2×10⁵ cells/ml in 24-well plates and cultured for 24 h, after which they were transfected with siRNAs targeting CYFIP2 and si-NC; the knockdown efficiency was verified 48 h after transfection by western blotting and reverse transcription-quantitative PCR (RT-qPCR). All the siRNAs were used at a final concentration of 5 nM. In addition, two NUAK2-specific short hairpin RNAs (shRNAs) (shRNA-NUAK2-1/shRNA-NUAK2-2) and a scrambled shRNA used as a negative control (shRNA-NC) were obtained from Shanghai GenePharma Co., Ltd. The sequences were as follows: shRNA-NUAK2-1, sense 5′-CCATAAGATCCTAGTGAAA-3′, antisense 5′-TTTCACTAGGATCTTATGG-3′; shRNA-NUAK2-2, sense 5′-GCATGACCATAAGATCCTA-3′, antisense 5′-TAGGATCTTATGGTCATGC-3′; shRNA-NC, sense 5′-GATCCCCTTCTCCGAACG-3′, antisense 5′-AGCTAAAAATTCTCCGAAC-3′. The corresponding shRNA oligonucleotides (synthesized by Guangzhou RiboBio Co., Ltd.) were cloned into the pLentiLox 3.7 lentiviral plasmid [American Type Culture Collection (ATCC)]. Lipofectamine 3000 was used to transfect 293T cells (ATCC) at a ratio of 3 µg lentiviral construct and 6 µg package mix [pLP1 (3): pLP2 (2): pLP/VSVG (3); (Invitrogen; Thermo Fisher Scientific, Inc.)] cultured at 37°C, according to the manufacturer's recommendations. The supernatants were collected at 48 and 72 h then mixed for ultracentrifugation (4°C, 72,000 × g, 2 h) to obtain lentiviral particles. The lentivirus (multiplicity of infection, 10) and HeLa cells were incubated in DMEM supplemented with 10% fetal bovine serum containing 8 µg/ml polybrene (MineBio Life Sciences Ltd.) at 37°C for 12 h and then washed with PBS. The medium was then replaced with fresh medium and cells were cultured for a total of 2 days prior to subsequent experiments.

The mRNA expression levels of NUAK2 and CYFIP2 were determined via RT-qPCR. Total RNA was extracted from tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using the PrimeScript™ RT kit (Takara Bio, Inc.) under the following conditions: 15 min at 37°C and 5 sec at 85°C. The synthesized cDNA was used as a template for PCR amplification. The reactions were performed using SYBR Green Taq Mix (Takara Bio, Inc.) in a Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.) under the following conditions: 45°C for 3 min, 95°C for 10 sec, 40 cycles at 95°C for 15 sec then 58°C for 1 min. Relative mRNA expression levels were quantified using the 2^−ΔΔCq method (15) and normalized to GAPDH. All RT-qPCR experiments were performed in triplicate. The primer sequences used were as follows: NUAK2, forward, 5′-TGAGAAACGACGGAGACAAGCTGCT-3′ and reverse, 5′-GTCTGGAGGTTTTGCTGCAGGTCTG-3′; CYFIP2 forward, 5′-TGGCGTCATCATTCCGTATCC-3′ and reverse, 5′-GTCAGGTCCTCACTCAAGC-3′; and GAPDH forward, 5′-TGTGTCCGTCGTGGATCTGA-3′ and reverse, 5′-CCTGCTTCACCACCTTCTTGA-3′.

The protein expression levels of NUAK2, CYFIP2, E-cadherin, N-cadherin, Snail and ZEB1 were determined via western blotting. Total protein was extracted from harvested cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 30 µg protein per lane was separated via SDS-PAGE on 15% gels and separated proteins were subsequently transferred to polyvinylidene fluoride membranes (EMD Millipore). Membranes were then blocked with 10% non-fat milk for 2 h at room temperature, and incubated with the following primary antibodies overnight at 4°C: Anti-NUAK2 (1:500; cat. no. ab107287; Abcam), anti-CYFIP2 (1:500; cat. no. sc-134308; Santa Cruz Biotechnology, Inc.), anti-E-cadherin (1:500; cat. no. sc-8426; Santa Cruz Biotechnology, Inc.), anti-N-cadherin (1:500; cat. no. sc-8424; Santa Cruz Biotechnology, Inc.), anti-Snail (1:500; cat. no. sc-271977; Santa Cruz Biotechnology, Inc.), anti-ZEB1 (1:500; cat. no. sc-10572; Santa Cruz Biotechnology, Inc.), anti-GAPDH (1:2,000; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.) and anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were washed with TBS-0.1% Tween-20 (TBST) for 5 min at room temperature. After three washes, the membranes were incubated at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.), HRP-conjugated goat anti-mouse IgG (1:2,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) or horseradish peroxidase (HRP)-conjugated mouse anti-goat IgG (1:2,000; cat. no. sc-2354; Santa Cruz Biotechnology, Inc.) for 2 h. After washing with TBST three times (10 min/wash), the protein bands were visualized using Western Blotting Luminescent Reagent (Santa Cruz Biotechnology, Inc.) and analyzed using ImageJ software (version 1.43; National Institutes of Health).

Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8) assay (cat. no. C0037; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, HeLa cells were seeded at a quantity of 1×10⁴ cells/well in 96-well plates and cultured for 6 h, after which they were incubated with 15 µl/well CCK-8 solution at 37°C for 24, 48 or 72 h. Subsequently, the optical density (OD) was calculated at a wavelength of 450 nm to determine cell proliferation.

To observe the expression of Ki67, IF staining was performed. HeLa cells were seeded onto glass coverslips in 6-well plates. Cells were incubated with lentiviruses containing shRNA-NUAK2 at 37°C for 2 days to silence NUAK2 gene expression. The cells were then incubated with si-CYFIP2 for an additional 36 h to obtain cells with low expression of NUAK2 and CYFIP2. After that, the cells were fixed with 4% paraformaldehyde (PFA; Millipore Sigma) at room temperature for 10 min, washed in PBS and blocked for 15 min in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime Biotechnology) at room temperature. After incubation with a primary antibody against anti-Ki67 (1:200; cat. no. sc-23900; Santa Cruz Biotechnology, Inc.) at 4°C overnight, cells were washed in PBS and incubated with a secondary antibody conjugated with Alexa Fluor 488 (1:1,000; cat. no. A32723; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Cells were then washed in PBS, incubated for 5 min with DAPI (1 µg/ml; cat. no. D1306; Invitrogen; Thermo Fisher Scientific, Inc.), and observations were performed using a fluorescence microscope (magnification, ×100; Olympus Corporation) and were analyzed with ImageJ version 1.43 software.

HeLa cells were seeded at 6×10⁴ cells/well in 6-well plates, cultured until they reached 80–90% confluence and were subjected to serum starvation for 4 h. Subsequently, a sterile 10-µl pipette tip was used to create a linear scratch in the cell monolayer, the cells were washed with PBS to remove debris, and the medium was replaced with serum-free DMEM/F12 1:1. Images were captured under a light microscope (Olympus Corporation) prior to and at 24 h after incubation.

HeLa cells (2×10⁴ cells/well) were cultured in the upper chamber of Transwell plates (8-µm pore size; Corning, Inc.). The surface of the upper chamber was pre-coated with Matrigel™ (BD Biosciences) at 37°C for 1 h. Complete medium (500 µl) supplemented with 10% bovine calf serum (Gibco; Thermo Fisher Scientific, Inc.) was added into the lower chamber. After 24-h incubation at 37°C, cells remaining on the upper membrane were wiped away, whereas cells that had invaded across the membrane were fixed with 4% PFA for 10 min at room temperature, stained with 0.1% crystal violet for 15 min at room temperature and counted under a light microscope.

The Pierce Co-IP Kit (cat. no. 26149; Thermo Fisher Scientific, Inc.) was used to explore the interaction between proteins, according to the manufacturer's protocol. Briefly, the HeLa cells cultured in a 10-cm plate were washed twice with pre-cooled PBS, and the PBS was discarded. Subsequently, 1 ml lysis buffer was used to fully lyse the cells, the lysate was transferred into 1.5-ml microcentrifuge tubes, centrifuged for 15 min (4°C, 14,000 × g), and the supernatant protein was taken for quantification. The total volume was made up to 500 µl with lysis buffer after 500 µg protein was taken. Subsequently, 30 µl Agarose A + G was used to pre-clear lysate at 4°C for 2 h. The pre-cleared lysate was centrifuged at 4°C for 5 min at 1,000 × g and the supernatant was removed and incubated overnight with shaking at 4°C by adding the corresponding antibody. The following antibodies were used: NUAK2 (1:10; cat. no. sc-374348; Santa Cruz Biotechnology, Inc.), CYFIP2 (1:50; cat. no. sc-134308; Santa Cruz Biotechnology, Inc.) and IgG (1:50; cat. no. sc-69786; Santa Cruz Biotechnology, Inc.), which was used as a control. After 12 h of antibody conjugation, Agarose A + G (30 µl/tube) was added and shaken at 4°C for 4 h. The supernatant was discarded after centrifugation at 1,500 × g for 5 min to obtain the sediment. The precipitate was then washed three times with pre-chilled PBS to obtain the protein sample for western blotting.

All experimental data are presented as the mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.). In order to assess whether data obtained from the tissue samples followed a normal distribution, a Shapiro-Wilk test was used. Based on this information, data were further analyzed using parametric tests. To compare differences among multiple groups, one-way ANOVA followed by Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.

UALCAN website predicted the expression levels of NUAK2 in CESC (Fig. 1A). The expression of NUAK2 was detected in CC tissues by RT-qPCR and western blotting. As shown in Fig. 1B and C, the protein and mRNA expression levels of NUAK2 were significantly increased in CC tissues compared with those in the control group. Similarly, NUAK2 exhibited higher expression in CC cell lines compared with that in Ect1 cells, with the maximum expression observed in HeLa cells (Fig. 1D). Therefore, HeLa cells were selected for subsequent experiments. These results indicated that NUAK2 was highly expressed in CC tissues and cell lines.

In order to determine the role of NUAK2 expression in CC, shRNA-NUAK2 was used. As shown in Fig. 2A and B, transfection of HeLa cells with shRNA-NUAK2-1 silenced NUAK2 expression more efficiently compared with shRNA-NUAK2-2; therefore, shRNA-NUAK2-1 was selected for subsequent experiments. As determined by CCK-8 assay, the OD value at 450 nm in the shRNA-NUAK2 group was significantly attenuated compared with that in the shRNA-NC group, whereas there were no changes between the untransfected control group and the shRNA-NC group (Fig. 2C). Furthermore, the protein expression levels of Ki67 were markedly decreased in the shRNA-NUAK2 group compared with those in the control group and the shRNA-NC group (Fig. 2D). These results indicated that knockdown of NUAK2 inhibited CC cell proliferation.

As shown in Fig. 3A-C, after 24 h of incubation, the scratches in the cell monolayer were wider and the relative cell migration rate was lower in the shRNA-NUAK2 group compared with that in the control group and the shRNA-NC group, suggesting that cell migration was inhibited when NUAK2 expression was knocked down. Similarly, the results of the Transwell invasion assay revealed that, compared with in the control and shRNA-NC groups, the invasive ability of HeLa cells was suppressed in the shRNA-NUAK2 group. Furthermore, western blotting revealed that NUAK2 knockdown elevated the expression levels of E-cadherin, and decreased the expression levels of N-cadherin, Snail and ZEB1 compared with those in the shRNA-NC group (Fig. 3D), suggesting that NUAK2 promoted EMT in CC cells. These data confirmed that NUAK2 knockdown suppressed the EMT of CC cells. Taken together, these data indicated that knockdown of NUAK2 expression decreased the mobility of HeLa cells after transfection.

The UALCAN website predicted the expression levels of CYEIP2 in CESC (Fig. 4A). To reveal the molecular mechanisms underlying the role of NUAK2 in CC, the STRING database was used to identify whether NUAK2 directly binds to CYFIP2. The results of western blotting (Fig. 4B) and RT-qPCR (Fig. 4C) revealed that CYFIP2 expression was decreased in tissues from patients with CC compared with that in the control group. Subsequently, the mRNA expression levels of CYFIP2 were detected in several CC cell lines (HeLa, C-33A, SiHa, CaSKi and HCC-94), which demonstrated that CYFIP2 was downregulated at different levels in these CC cell lines compared with that in Ect1 cells (Fig. 4D). To verify the targeted binding of NUAK2 and CYFIP2, Co-IP was performed. The results of Co-IP demonstrated that NUAK2 could interact with CYFIP2 (Fig. 4E). The western blotting results confirmed that NUAK2 knockdown increased the expression levels of CYFIP2 (Fig. 4F). These results indicated that CYFIP2 was expressed at low levels in CC and was negatively associated with NUAK2.

In order to further explore the role of CYFIP2 in CC, si-CYFIP2 was used in the following experiments. The results presented in Fig. 5A and B show that both si-CYFIP2-1 and si-CYFIP2-2 could reduce the protein and mRNA expression levels of CYFIP2 in HeLa cells, but si-CYFIP2-2 was more efficient. Thus, si-CYFIP2-2 was selected to knock down CYFIP2 expression. The CCK-8 assay and IF revealed that si-CYFIP2 could enhance cell proliferation compared with that in the shRNA-NUAK2+ si-NC group (Fig. 5C and D). These results suggested that the effect of NUAK2 knockdown on CC cell proliferation may be reversed by inhibition of CYFIP2 expression.

As presented in Fig. 6A-C, in HeLa cells transfected with shRNA-NUAK2, transfection with siRNA to interfere with CYFIP2 expression could accelerate gap closure in the cell monolayer and enhanced the invasive ability of HeLa cells compared with in the shRNA-NUAK2 group and the shRNA-NUAK2+ siRNA-NC group. In addition, inhibition of CYFIP2 expression reversed the effect of NUAK2 knockdown on the EMT phenotype of HeLa cells (Fig. 6D). These results demonstrated that interference with NUAK2 inhibited CC cell migration and the EMT process through upregulation of CYFIP2.