Cervical cancer tissue and adjacent tissue samples were obtained from 40 female patients (age, 43.57±2.79) with cervical cancer who attended Jingmen First People's Hospital between January 2018 and May 2019. The tissue samples were maintained in liquid nitrogen and stored at −80°C. Exclusion criteria: i) other gynaecological diseases; ii) severe internal and external diseases; iii) history of chemoradiotherapy; and iv) history of pelvic organ surgery. All research subjects provided written informed consent and the study was approved by the Ethics Committees of Jingmen No. 1 People's Hospital.

The human endometrial epithelial cell (Ect1/E6E7) line were purchased from American Type Culture Collection. The cervical cancer cell lines (HeLa, C-33 A, SiHa and ME-180) were purchased from the American Type Culture Collection. All the cells were cultured in DMEM (cat. no. D0819; Sigma-Aldrich; Merck KGaA), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ atmosphere.

Among the cervical cancer cell lines, as miR-802 was expressed at the lowest levels in SiHa cells, SiHa cells were used in the following experiments. For transfection, 2 ml cell solution, containing 1×10⁶ cells/well and culture medium, was plated into 6-well plates and incubated at 37°C in a 5% CO₂ atmosphere until the cell confluence reached 40–60%. To prepare A transdye, 20 pmol miR-802 mimic (forward, 5′-UCAGUAACAAAGAUUCAUCCUUGU-3′ and reverse, 5′-ACAAGGAUGAAUCUUUGUUACUGA-3′; Shanghai GenePharma Co., Ltd.), the negative control for the mimic (NC-mimic; a non-targeting sequence; forward, 5′-UUUUACUACACAAAAGUACUG-3′ and reverse, 5′-CAGUACUUUUGUGUAGUACAAA-3′; Shanghai GenePharma Co., Ltd.), BTF3 overexpression plasmid (pWZL-BTF3; Shanghai GenePharma Co., Ltd.) or the empty plasmid (pWZL-Blast; pWZL-NC; Shanghai GenePharma Co., Ltd.) were dissolved in 50 µl DMEM (Hyclone; GE Healthcare Life Sciences) and fully mixed. To prepare B transdye, 1 µl Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was dissolved in 50 µl DMEM, incubated for 15 min at room temperature and then mixed with A transdye. This solution (50 µl) was subsequently added into each well of the plate and incubated at 37°C with 5% CO₂. Following 24 h of transfection, the medium was changed and the cells were collected after culture for 72 h.

To investigate the effects of miR-802 on the viability, migration and invasion of cervical cancer cells, the cells were divided into three groups: Control (untreated cells), NC-mimic (cells transfected with the NC-mimic) and miR-802 mimic (cells transfected with the miR-802 mimic) group. To determine the effects of BTF3 on the viability, migration and invasion of cervical cancer cells, the cells were divided into five groups: Control (untreated cells), pWZL-NC + NC-mimic (cells co-transfected with empty plasmid and NC-mimic), pWZL-BTF3 + NC-mimic (cells co-transfected with BTF3 overexpression plasmid and NC-mimic), pWZL-BTF3 + miR-802 mimic (cells co-transfected with BTF3 overexpression plasmid and miR-802 mimic) and miR-802 mimic (cells transfected with miR-802 mimic) group.

The target-binding region of miR-802 and BTF3 was predicted using TargetScan (version 7.2; www.targetscan.org/vert_72/). For the dual-luciferase reporter assays, the 3′-UTR of BTF3 containing miR-802 binding sites was inserted into a pmirGLO dual-luciferase vector (Promega Corporation) to generate wild-type (WT) BTF3-3′-UTR. The mutant (mut) 3′ UTR of BTF3 was synthesized using a Site-Directed Mutagenesis kit (Thermo Fisher Scientific, Inc.) and inserted into the pmirGLO dual-luciferase vector to generate BTF3-3′-UTR-mut. The pmirGLO vector containing WT or mut BTF3 3′-UTR was co-transfected with the miR-802 mimic or NC-mimic into SiHa cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 48 h, the relative luciferase activity in the cells was measured using a Dual-Luciferase Reporter Assay kit (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activities were normalized to Renilla luciferase activities.

Following 24 h of transfection, SiHa cells were resuspended in DMEM without FBS until the cell density reached 1×10⁶ cells/ml. Then, 100 µl cell suspension was plated into the upper chambers of the Transwell plate (Corning, Inc.) and cultured at 37°C for 6 h. DMEM with 10% FBS was plated into the lower chamber. Following the incubation, the non-migratory cells were removed with a cotton swab and the migratory cells were washed three times with PBS, and fixed with 4% formaldehyde (cat. no. P0099; Beyotime Institute of Biotechnology) for 20 min at room temperature and with 1% Triton X-100 (cat. no. P0096; Beyotime Institute of Biotechnology) for 5 min at room temperature. After washing the cells three times with PBS, the cells were stained with hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology) for 20 min at room temperature and then washed under running water. Stained cells were counted in five randomly selected fields using a light microscope (magnification, ×200; Olympus Corporation); the mean number of cells per field of view was calculated.

Following 24 h of transfection, SiHa cells were resuspended in DMEM without FBS until the cell density reached 1×10⁶ cells/ml. The upper chambers of the Transwell plates (pore size, 8.0 µm; Corning, Inc.) were precoated with 50 µl Matrigel (BD Biosciences) for 2 h at 4°C. Then, the cell suspension was subsequently plated into the upper chamber of the Transwell plate. DMEM, supplemented with 10% FBS was plated in the lower chambers. Following incubation for 24 h at 37°C, the invasive cells in the lower chamber were fixed in 5% gluteraldehyde for 30 min at room temperature and stained with 0.5% crystal violet for 20 min at room temperature, while the non-invasive cells remaining in the upper chamber were removed using a cotton swab. Stained cells were observed in three randomly selected fields of view using a light microscope (magnification, ×200).

SiHa cells (2×10⁴ cells/well) were plated into 96-well plates in DMEM containing 10% FBS and cultured for 24 h at room temperature. Subsequently, 10 µl Cell Counting Kit-8 (cat. no. 96992; Sigma-Aldrich; Merck KGaA) solution was added into each well, according to the manufacturer's protocol, and incubated for 4 h at 37°C. The absorbance at 450 nm was used a Multiskan GO microplate reader (Shanghai Bajiu Industrial Co., Ltd.). The experiment was conducted in triplicate and the average absorbance value was calculated.

SiHa cells were washed twice with cold PBS and total protein was extracted from the cells using RIPA lysis buffer (Cell Signaling Technology, Inc.). Total protein was quantified using the bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). The extracted protein was denatured for 5 min at 100°C, and 50 µg/µl protein/lane was separated via 15% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% milk at room temperature for 1 h. The membranes were incubated with the following primary antibodies at 4°C overnight: Anti-N-cadherin (1:1,000; cat. no. ab18203; Abcam), anti-BTF3 (1:1,000; cat. no. ab203517; Abcam), anti-matrix metallopeptidase (MMP)9 (1:1,000; cat. no. ab73734; Abcam), anti-MMP2 (1:1,000; cat. no. ab37150; Abcam), anti-E-cadherin (1:1,000; cat. no. ab40772; Abcam) and anti-GAPDH (1:2,000; cat. no. ab8245; Abcam). Following the primary antibody incubation, the membranes were incubated with goat anti-mouse or goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibodies (cat. nos. SA00001-1 and SA00001-2; 1:2000; ProteinTech Group, Inc.) for 2 h at room temperature and then washed with PBS three times. Protein bands were visualized using an ECL western blotting kit (cat. no. 93-K820-500; Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.) and the expression levels were semi-quantified using ImageJ v4.7 software (National Institutes of Health).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the concentration of RNA was measured using a NanoDrop™ spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.), which was then diluted to 500 ng/µl. Total RNA was reverse transcribed into cDNA using a SuperScript™ II First-Strand cDNA synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using a QuantiFast SYBR Green PCR kit (cat. no. 204057; Qiagen, Inc.) according to the manufacturer's protocol. The following thermocycling conditions were used for the qPCR: Initial denaturation at 94°C for 2 min; 35 cycles of 94°C for 30 sec, 63°C for 30 sec, 72°C for 1 min; and a final extension at 72°C for 7 min, prior to being maintained at 4°C. The following primer sequences used are listed in Table I. Expression levels were quantified using the 2^−ΔΔCq method (21) and normalized to the loading controls, GAPDH or U6, for mRNA or miR-802 expression, respectively.

Statistical analysis was performed using GraphPad Prism v6.01 software (GraphPad Software, Inc.) and data are presented as the mean ± SD of ≥3 independent experiments. Statistical differences between two groups were determined using a paired Student's t-test, whereas statistical differences among multiple groups were analyzed using a one-way ANOVA, followed by a Bonferroni's correction post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-802 were significantly decreased in the cervical cancer tissues compared with the adjacent tissue (P<0.001; Fig. 1A). Similarly, the expression levels of miR-802 were observed to be significantly decreased in the cervical cancer cell lines (HeLa, C-33 A, SiHa and ME-180) compared with the EEC cell line (P<0.001; Fig. 1B).

The expression levels of miR-802 were significantly increased following the transfection of the miR-802 mimic into SiHa cells compared with the control and NC-mimic groups (P<0.001; Fig. 2A). However, following the overexpression of miR-802 in the cells, no significant difference was observed in the cell viability compared with the control group (P>0.05; Fig. 2B). Moreover, a Transwell assay was performed to determine the migratory ability of SiHa cells; the results revealed that the overexpression of miR-802 significantly decreased the migratory rate compared with the control and NC-mimic groups (P<0.001; Fig. 2C and D). Similarly, the cell invasive ability was significantly inhibited in the miR-802 mimic-transfected cells compared with the control and NC-mimic groups (P<0.001; Fig. 2E and F).

The possible target genes of miR-802 were predicted using TargetScan software and it was discovered that miR-802 bound to the 3′-UTR of BTF3 (Fig. 3A). Subsequently, two pmirGLO dual-luciferase reporter vectors, namely, BTF3-3′-UTR and BTF3-3′-UTR-mut, were constructed. BTF3-3′-UTR or BTF3-3′-UTR-mut were co-transfected with the miR-802 mimic into the cells. The miR-802 + BTF-3′-UTR group displayed significantly decreased relative luciferase activity compared with the other two groups: therefore, the results indicated that BTF3 was a direct target gene of miR-802 (P<0.001; Fig. 3B). The expression levels of BTF3 in the pWZL-BTF3 group were significantly increased compared with the pWZL-NC and control groups, suggesting that the BTF3 overexpression transfections were successful (P<0.001; Fig. 3D). Furthermore, the expression levels of miR-802 were significantly increased in the pWZL-BTF3 + miR-802 mimic group compared with the pWZL-BTF3 + NC-mimic group (P<0.001; Fig. 3C). The highest expression levels of BTF3 were observed in the pWZL-BTF3 + NC-mimic group, whilst the expression levels of BTF3 in the pWZL-BTF3 + miR-802 mimic group were significantly decreased compared with the pWZL-BTF3 + NC-mimic group, but significantly increased compared with the miR-802 mimic group (P<0.001; Fig. 3E-G).

The pWZL-BTF3 + NC-mimic group significantly increased cell viability compared with the pWZL-NC + NC-mimic group; however, co-transfection with the miR-802 mimic (pWZL-BTF3 + miR-802 mimic group) significantly reversed the effect of BTF3 on cell viability (P<0.001; Fig. 4A). Moreover, the overexpression of BTF3 significantly increased the cell migratory rate compared with the pWZL-NC + NC-mimic group, whereas this effect was significantly inhibited following co-transfection with the miR-802 mimic (P<0.001; Fig. 4B and C). Similarly, co-transfection with the miR-802 mimic significantly suppressed the function of BTF3 alone in promoting the cell invasive ability (P<0.001; Fig. 4D and E). In addition, the regulatory effect of miR-802 on the EMT process of cervical cancer cells was investigated. The results demonstrated that the pWZL-BTF3 + NC-mimic group displayed significantly increased mRNA and protein expression levels of MMP2, MMP9 and N-cadherin compared with the pWZL-NC + NC-mimic group, whist inhibiting the expression levels of E-cadherin expression; however, the co-transfection of cells with pWZL-BTF and miR-802 mimic significantly reversed the effects of BTF3 on these genes (P<0.001; Fig. 4F-H).