In the present study, paraffin sections were collected from 33 patients with NC, 33 patients with LSIL, 40 patients with HSIL and 45 patients with CC among patients (47.51±11.41 years) treated at Feicheng People's Hospital, (Tai'an, China), between January 2010 and December 2019. The tissues were cut into 4-µm sections and 0% neutral buffer formalin solution was used to fix the samples at room temperature for 24–48 h. The clinical stage was determined according to the 2018 International Federation of Gynecology and Obstetrics (FIGO) staging guidelines (4). Patient age, previous human papilloma virus (HPV) test results, thyrocalcitonin levels, colposcopy findings, and pathology findings were recorded. For each tissue collection, a written consent from the subject was obtained. The present study was approved by the Ethics Committee of Qilu Hospital (approval no. KYLL-2017-560).

Human CC cell lines (C33A, SiHa, HeLa, CaSKi, ME-180 and HH-8) and HUCEC cells (normal human cervical epithelial cells), which were used as a control, were provided by Shanghai Meixuan Biotechnology Co., Ltd. Cells were cultured in 90% Dulbecco's Modified Eagle's Medium (DMEM) (high glucose; H) + 10% foetal bovine serum (FBS) (Hyclone; Cytiva). Cells were transferred under sterile conditions, seeded into 8–10 ml of complete medium (90% DMEM [H] + 10% FBS), and cultured at 37°C and 5% CO₂ in an incubator. The SiHa and HeLa cell lines, which have high FLNa and UCP2 expression levels, were selected for the subsequent experiments.

The following antibodies were used for immunofluorescence staining: Anti-GAPDH (1:5,000; cat. no. Ab8245; Abcam); anti-UCP2 (1:100; cat. no. 11081-1-AP; ProteinTech Group, Inc.) and anti-FLNa (1:100; cat. no. ab76289; Abcam). The antibodies were used to detect UCP2 and FLNa expression in different CC cell lines.

The following antibodies were used for immunohistochemical staining: Anti-UCP2 (1:200; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:200; cat. no. ab76289; Abcam), anti-P16 (1:200; cat. no. 10883-1-AP; ProteinTech Group, Inc.), and anti-Ki67 (1:1,000; cat. no. ab92742; Abcam).

The following primary antibodies were used for western blotting of relevant proteins: Anti-UCP2 (1:1,000; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:1,000; cat. no. ab76289; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), anti-ERK1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-P-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-AKT1 (1:1,000; cat. no. ab227100; Abcam), anti-P-AKT1 (1:10,000; cat. no. ab81283; Abcam), and anti-BCL-2 (1:1,000 cat. no. ab32124; Abcam).

After the cell samples were fully lysed using Lysis Buffer (Beyotime Institute of Biotechnology), they were centrifuged at 12,000 × g, 4°C for 5 min, and part of the supernatant was collected for protein determination using the bicinchoninic acid (BCA) method.

For the western blotting, the protein was separated via SDS-PAGE (10 and 12% gels), and the concentration was adjusted to 1 mg/ml, the protein quality was 20 µg, and the volume was 20 µl per lane. Before separating the proteins on a gel, a standard curve was drawn according to the optical density (OD) value and the standard protein concentration to calculate the total protein concentration in the sample. After transferring the sample solution, the membrane was blocked at room temperature for 2 h., using 1% BSA (Roche Diagnostics). The membrane was removed and then washed with tris-buffered saline Tween-20 (TBST) on a shaking table for 5 min, 3 times. In an incubation bag, the primary antibodies (FLNa and UCP2) were diluted with sealing solution and incubated overnight at 4°C. The film was washed with TBST for 5 min, 3 times, and then the sheep anti-rabbit secondary antibody (1:5,000; cat. no. SA00001-2 Jackson ImmunoResearch Laboratories, Inc.) horseradish peroxidase-conjugated was added and BCA Protein Quantitation kit (Beyotime Institute of Biotechnology) was used.

The SiHa and HeLa cells were blocked with 3% BSA-PBS (Roche 735094) for 30 min at room temperature. The present study used anti-UCP2 and anti-FLNa as primary antibodies and FITC [Fluorescein (FITC)-conjugated Affinipure Goat Anti-Rabbit IgG; 1:50; cat. no. SA00003-2; ProteinTech Group, Inc.] as a secondary antibody. The cell samples were fixed in 4% precooled paraformaldehyde at room temperature for 15 min, washed with phosphate-buffered saline (PBS) 3 times (3 min each time), and incubated in 0.5% Triton X-100 for 20 min at room temperature. Next, diluted antibody solution (in PBS containing 5% bovine serum albumin) (Hyclone; Cytiva) was added (PBS 0.01 M, pH 7.4 was used in the blank control group), and the samples were incubated at 4°C overnight. On the next morning, the samples were removed from the refrigerator, placed at room temperature for 15 min, and then washed with PBS (0.01 M, pH 7.4) 5 times (5 min each). After removal of any extra PBS, FITC was added, and then DAPI was added, followed by incubation in the dark for 2 min. The nucleus was identified by blue fluorescence microscope (Olympus Corporation; ×10 magnification).

Antigen retrieval was performed using TE buffer pH 9.0, heating in the microwave on band 3 for 10–15 min (700 W oven), the retrieval solution was allowed to cool at room temperature. Endogenous peroxidase activity was blocked by incubating sections in 3% H₂O₂ solution in ddH₂O for 10 min at room temperature. Tissue sections (4 mm-thick) were stained with FLNa antibody (diluted in PBS, 1:200) and UCP2 antibody (diluted in PBS, 1:200). The expression of FLNa and UCP2 was evaluated in cancer cells. (DAB Detection Kit, Ventana Medical Systems, Inc)The expression scores were grouped as follows: 0, <10% positive cells; 1, 10–25% positive cells; 2, 26–50% positive cells; 3, 51–75% positive cells; and 4, >75% positive cells. The cells were stained with DAB for 40 sec and haematoxylin and eosin for 8 min, both at room temperature, and the staining intensity was rated as 0, none; 1, weak; 2, mild; and 3, strong. The final score was the product of the quantitative expression score and staining intensity and was denoted as negative (0–1) or positive (≥2). All immunohistochemical results were reported by the pathologists at Feicheng People's Hospital (Tai'an, China) and reviewed by another experienced pathologist. A confocal microscope was used to view the images (Olympus Corporation; ×40 and ×200 magnification).

The primers (Table I) were designed and synthesized by Shanghai Meixuan Biotechnology Co., Ltd. TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract the total RNA from SiHa and HeLa cells, which was stored at −80°C. The total RNA (2 µg) was used for the synthesis of cDNA according to a PrimeScript RT Reagent kit with g DNA Eraser. Reverse transcription was performed as 50°C for 10 min). The fluorophore was provided by Shanghai Meixuan Biotechnology Co., Ltd. (SYBR Premix Ex Taq). The PCR conditions were predenaturation at 95°C for 30 sec, then 45 cycles of 95°C for 5 sec and 60°C for 30 sec. The data were analysed using the 2^−ΔΔCq relative-expression method (28).

Small interfering RNA (siRNA) sequences were designed and synthesized by Shanghai Meixuan Biotechnology Co., Ltd. Once the cells reached 40–50% confluency, the cells were seeded into 24-well plates and transfected with Lipofectamine^® 2000 according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). First, 20 pmol siRNA was dissolved in 50 µl Opti-MEM culture medium with free FBS. Then, 1 µl Lipofectamine^® 2000 was dissolved in 50 µl Opti-MEM culture medium for 5 min at room temperature. Finally, the aforementioned two solutions were mixed while resting for 20 min at room temperature, then 400 µl mixed solution was added to each well in the 24-well plates, and the transfection medium was changed to cultured medium (Hyclone; Cytiva) containing 90% DMEM high sugar and 10% FBS) after 4–6 h. The cells were extracted for the PCR experiment after 48 h. siRNAs were transfected into cells with Lipofectamine^® 2000 for 48 h, and then cells were collected for qPCR to analyse the mRNA levels of UCP2 and FLNa. After 48 h, a sequence with good interference was selected for later experiments; a third siRNA was selected to knockdown UCP2, and a second siRNA was selected to knockdown FLNa in HeLa and SiHa cells. Each in vitro experiment was performed three independent times and in three replicates.

Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology) was used to detect cell survival. The absorbance at 450 nm was measured with a microplate reader (Multiskan 3; Thermo Fisher Scientific, Inc.). The measurement was 5×10³/100 µl for each sample. All experiments were repeated at least three times. The equation used for the calculation of the cell proliferation inhibition rate was as follows: Inhibition ratio=(OD value of control group-OD value of experimental group)/(OD value of control group-OD value of blank hole) ×100%.

The transfected cells were digested and seeded into 6-well plates. Once the cells reached 100% confluency, the medium was replaced with serum-free medium to starve the cells overnight. Different siRNAs were transfected into different groups of cells, and the medium was replaced with fresh serum-free medium after 4–6 h. A 100-µl pipette tip was used to scratch the plates, an image of which was captured and recorded as 0 h. After 48 h, another image was captured to analyse cell confluency relative to that at 0 h. The present study used Image Pro Plus software (version 6.0; Media Cybernetics, Inc.) to test and calculate the distance between the cells. The formula used for the calculation of the cell migration distance was as follows: Cell migration distance=0 h intercellular distance-48 h intercellular distance. A confocal microscope was used to detect the cells (Olympus Corporation; ×100 magnification).

A total of 5×10⁴ cells were inoculated into a Transwell chamber (BD Biosciences), and 500 µl of fresh medium containing 20% FBS was added to the lower chamber. The chamber was incubated at 37°C and 5% CO₂ for 48 h. Next, the cells were fixed in 4% paraformaldehyde for 10 min, stained with crystal violet solution for 5 min at room temperature and washed with water until the base membrane was transparent. Lastly, images of the cells were captured using a confocal microscope (Olympus Corporation; ×100 magnification), then the cells were counted and analysed.

The transfected cells were washed twice using precooled PBS and then digested in 70% alcohol at 4°C overnight. For cell cycle detection, a cell cycle detection kit was obtained from Beyotime Institute of Biotechnology. A FACSCalibur flow cytometer (BD Biosciences) was used to detect red fluorescence at an excitation wavelength of 488 nm. Appropriate analysis software (FlowJo; version 10; FlowJo LLC) was used for DNA content and light scattering analyses. For apoptosis detection, an Annexin V-FITC apoptosis assay kit was obtained from Beyotime Institute of Biotechnology. Cells were collected and treated as described in the kits and analysed by flow cytometry.

SPSS software (version 21.0; IBM Corp.) was used for immunohistochemical staining statistical analysis. The χ² and Fisher's exact test were performed. For the cell line experiments, all assays were performed in at least triplicate. The results are expressed as the mean ± SD and analysed by unpaired t-test or one-way ANOVA followed by Tukey's post hoc test, with SPSS software (version 21.0). P<0.05 was considered to indicate a statistically significant difference.

To detect the expression levels of FLNa and UCP2 in CC, the present study performed immunohistochemical staining on 33 NC, 33 LSIL, 40 HSIL and 45 CC tissues. Representative images are presented in Fig. 1A. The expression levels of FLNa and UCP2 in CC were rated as the product of the quantitative score and staining intensity (n=45). The scores ranged from 0 to 12, and scores of 0–1 were rated as negative, whereas scores ≥2 were rated as positive. The median score of each protein in the tissues was used as the threshold to separate low expression from high expression (Table II). The results show that FLNa and UCP2 were highly expressed in the CC tissues (Table III). The FLNa and UCP2 expression levels were detected by PCR and western blotting in six human CC cell lines (C33A, SiHa, HeLa, CaSKi, ME-180 and HH-8), and HUCEC cells (normal human cervical epithelial cells) as control cells. All experiments were repeated at least three times (Fig. 2A-C). The SiHa and HeLa cell lines, which had the highest FLNa and UCP2 expression levels, were selected for the subsequent experiments. To investigate the expression of FLNa/UCP2 in cells, the present study performed immunofluorescence staining in SiHa and HeLa cells. The immunofluorescence quantitative analysis is presented in (Fig. 2D). It was revealed that FLNa and UCP2 were mainly located in the cytoplasm of CC cells, as determined by immunofluorescence staining (Fig. 2D). According to the results of the present study, FLNa and UCP2 were highly expressed in CC cells. The immunohistochemical staining experiment showed the same results, and thus, SiHa and HeLa cells were selected for the subsequent experiments in order to determine the function of FLNa/UCP2 in CC cell lines.

To investigate the associations between FLNa/UCP2 expression in CC tissues and clinicopathological factors, the present study analysed age, tumour size, tumour differentiation, clinical stage, lymph node metastasis and histological type of the patients. No significant association was observed between the expression levels of FLNa in CC and age (P=0.714), tumour size (P=0.064), tumour differentiation (P=0.317), clinical stage (P=0.828), lymph node metastasis (P=0.737) or histology (P=0.060) (all P>0.05). No significant association was observed between the expression level of UCP2 in CC and age (P=0.276), tumour size (P=0.194), tumour differentiation (P=0.064) or histology (P>0.999). However, significant associations were observed for clinical stage (P=0.009) and lymph node metastasis (P=0.022) (both P<0.05) (Table IV).

To investigate whether the predictive function of FLNa/UCP2 was better than that of p16 and Ki-67 in cervical HSIL progression to CC, the present study stained for FLNa, UCP2, P16 and Ki-67 as four CC markers in immunohistochemical staining. The expression levels of these four markers were observed under ×200 and ×40 magnification under a confocal microscope (Olympus Corporation). FLNa and UCP2 were primarily stained in the cytoplasm of CC cells as brown or dark-brown particles. The positive rate of FLNa was 86.7% (39/45), and the positive rate of UCP2 was 22.2% (10/45). p16 was expressed in the nucleus as brown or dark-brown particles, and the positive rate was 97.8% (44/45). Ki67 was expressed in the nucleus as brown or dark-brown particles, and the positive rate was 100% (45/45). Representative images are presented in Fig. 1B. The present study compared the positive rates of FLNa, UCP2, p16 and Ki67 between the CC and HSIL tissues (Table V). Significant differences in FLNa (P=0.005) and UCP2 (P=0.023) were observed between HSIL and CC tissues. No significant difference was observed in p16 (P=0.917) or Ki67 (P=0.471). FLNa and UCP2 were superior to p16 and Ki67 for early prediction of progression from HSIL to CC. The present study also analysed the associations between the expression levels of FLNa, UCP2 and p16 in CC and HSIL tissues (Table VI). It was revealed that in the CC tissues, both FLNa and p16 were positively expressed in 39 cases, and neither was expressed in one case (P<0.01). Both UCP2 and p16 were positively expressed in 10 cases, and neither was expressed in one case, indicating a positive association, though this result was not statistically significant (P>0.05). In HSIL tissues, both FLNa and p16 were positively expressed in 29 cases, and neither were expressed in two cases, but this was not statistically significant (P>0.01). Both UCP2 and p16 were positively expressed in two cases, and neither was expressed in two cases, but this result was not statistically significant (P>0.05). The aforementioned analysis indicated that there is an association between the expression levels of FLNa and p16 (P<0.01).

To investigate the functions and signalling pathways of FLNa and UCP2 in the development and progression of CC, FLNa and UCP2 siRNAs (Table VII) were designed and synthesized to downregulate the expression of FLNa and UCP2 via plasmid transfection, and the knockdown effect was confirmed via western blotting HeLa and SiHa cells with stable, high expression levels of FLNa and UCP2 were used for subsequent experiments. All experiments were repeated at least three times. Based on the qPCR, a third siRNA was selected to knockdown UCP2, and a second siRNA was selected to knockdown FLNa in HeLa and SiHa cells (Fig. 3A and B). To investigate the functions and mechanisms of action underlying UCP2 and FLNa, UCP2 and FLNa expression was knocked down in HeLa and SiHa cells, and the CCK-8 assay was used to analyse cell proliferation. The present study used the cellular proliferation inhibition rate to present the results of the CCK-8 assays, which demonstrated that cell proliferation was inhibited (Fig. 3C). The present study also used flow cytometry to analyse cell apoptosis, which revealed that following UCP2 and FLNa knockdown in HeLa and SiHa cells, there was no significant difference in apoptosis compared with the control groups (Fig. 4A). Following UCP2 and FLNa knockdown in HeLa cells and UCP2 knockdown in SiHa cells, more cells were arrested at the G₂ phase. Following UCP2 knockdown in SiHa cells G2 cells content increased. Following FLNa knockdown in SiHa cells, no significant cell cycle arrest was observed (Fig. 4B).

The scratch test and Transwell assay were used to detect cell migration and invasion. The results revealed that cell migration (Fig. 3D) and cell invasion were significantly decreased (Fig. 3E) with knockdown of these two proteins.

Western blotting was performed in order to detect the expression levels of relevant proteins (Fig. 5A), including ERK, p-ERK, AKT, p-AKT and Bcl-2. The results showed no significant change in ERK/p-ERK, AKT1 or Bcl-2 levels (Fig. 5D), but p-ERK1/2 (Fig. 5B) and p-AKT1 (Fig. 5C) were downregulated. PCR showed that the mRNA levels of Ras (Fig. 5E), MMP-2 (Fig. 5F) and MMP-9 (Fig. 5G) were decreased.