The cervical cancer cell lines ME-180 (cat. no. HTB-33™), HT-3 (cat. no. HTB-32™), HeLa (cat. no. CCL-2™) and SiHa (cat. no. HTB-35™) (ATCC; Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. Human cervical epithelial cells (HCerEpiC; ScienCell Research Laboratories, Carlsbad, CA, USA; cat. no. CP7060) (27), were cultured in cervical epithelial cell medium (ScienCell Research Laboratories). Cells were cultured in an atmosphere of 5% CO₂ at 37°C.

Cervical tumor samples (n=10, 40–50 years of age) were collected in the Fourth People's Hospital of Shaanxi from women with the average year of 42 years undergoing hysterectomies without having been treated with radiotherapy or chemotherapy. Two patients had a diagnosis of squamous cell carcinoma (SCC), 6 presented with adenomatous carcinoma (ADC) and 2 patients had an intermediate diagnosis of adenosquamous cell carcinoma (ASC). Normal tissues (n=10, 40–50 years of age) were collected in the Fourth People's Hospital of Shaanxi from women with the average year of 45 years old undergoing surgery for myoma or adenomyoma. Normal tissue samples were non-malignant and negative for human papilloma virus and ThinPrep cytologic tests. The collections were performed between August 2017 and September 2017 and were approved by the Ethics Committee of The Fourth People's Hospital of Shaanxi. Informed consent was obtained from all of the patients.

Total RNA of cells was extracted by using TRizol (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and reverse-transcribed into cDNA using the PrimeScript™ First Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China). The volume of reverse-transcription polymerase chain reaction (RT-qPCR) was 20 µl and it contained 10 µl SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.). The primers were as follows: HOXA5, sense primer: 5′-AGCCACAAATCAAGGACACA-3′ and antisense primer: 5′-GCTCGCTCACGGAACTATG-3′; GAPDH, sense primer: 5′-CGAAGGTGAAGGTCGGAGT-3′ and antisense primer: 5′-GAAGATGGTGATGGGATTTC-3′. The cycling parameters consisted of 94°C for 30 sec; 40 cycles of 94°C for 40 sec, 60°C for 30 sec and 72°C for 30 sec and 72°C for 10 min. The relative levels of gene expression were quantified using the 2^−ΔΔCt method (28). GAPDH was the reference gene. The experiment was repeated three times.

Cells were treated with lysate and protein was extracted. Then, the protein was quantified with a BCA kit (Beyotime Institute of Biotechnology, Nantong, China). After loading 25 µg of the protein onto gel for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were separated and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, USA) by electrophoretic transfer. The PVDF membranes were then incubated with 5% skim milk for 1.5 h, followed by incubation overnight at 4°C with the primary antibodies (all from Abcam Inc., Cambridge, MA, USA): anti-HOXA5 (dilution 1:800; cat. no. ab82645) anti-AKT (dilution 1:500; cat. no. ab8805), anti-Akt (phospho S473) (dilution 1:600; cat. no. ab81283), anti-p27 (dilution 1:1,000; cat. no. ab32034) and anti-GAPDH (dilution 1:500; cat. no. ab9485). Next, the polyvinylidene fluoride was incubated with a secondary antibody (dilution 1:800; cat. no. ab7090) diluted in the blocking buffer. Finally, the protein was detected using enhanced chemiluminescence. The experiment was repeated three times.

The full-length HOXA5 gene (GenBank™ accession number NM_019102.3) was amplified using RT-PCR, followed by insertion into the plasmid pcDNA.3.1 (Clontech Inc., Palo Alto, CA, USA). The restriction enzymes used for the cDNA and plasmid pcDNA.3.1 were EcoRI and BamHI. The recombinant plasmid was then transducted into Escherichia coli DH5α cells (Tiangen, Beijing, China), followed by amplification at 37°C. Afterwards, the recombinant plasmids were extracted using a plasmid DNA extraction kit (Takara Biotechnology Co., Ltd.). The recombinant plasmid containing the correct sequence was named pcDNA.3.1-HOXA5.

The ME-180 and HT-3 cell lines were separately cultured in 96-well plates and incubated in an incubator (Thermo Fisher Scientific, Inc.) with 5% CO₂ for 24 h. The cells were transfected with pcDNA.3.1-HOXA5 (0.2 µg), pcDNA.3.1 (0.2 µg) or pcDNA.3.1-p27 (0.2 µg) using Turbofect (0.5 µl; Thermo Fisher Scientific, Inc.) and incubated at 37°C in 5% CO₂ for 24, 36, 48 or 72 h. Transfection efficiency was measured by RT-qPCR and western blot assays.

Cell apoptosis was examined using an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit (Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the supplier's instructions. Briefly, cells were harvested and centrifuged at 200 × g for 5 min. The cells (5×10⁵ cells/ml) were resuspended in the binding buffer and mixed with 5 µl of Annexin V-FITC in 195 µl of the cell suspension for 30 min at 4°C. Afterwards, cells were incubated with PI (6 µl) for 8 min. The apoptotic cells were quantified by FACS analyzer (Beckman Coulter, Brea, CA, USA). The experiment was repeated three times.

Caspase-3 activity in the cells was measured in line with the explanatory memorandum of a caspase-3 activity assay kit (Beyotime Institute of Biotechnology). In brief, cells (1×10⁶) were centrifugated, washed trice with PBS and incubated in 500 µl lysis buffer on ice for 15 min. The activity of caspase-3 was tested by a 200 mmol/l AcDEVD-MCA fluorogenic substrate in the assay buffer, and fluorescence intensity was quantified with spectrofluorometry. The experiment was repeated three times.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantify cell viability according to the specifications. Firstly, cells were incubated in a 96-well plate in humid atmosphere of 5% CO₂ at 37°C for 24 h followed by transfection. MTT (5 g/l) diluted in phosphate-buffered saline (PBS) was added and incubated at 37°C for 6 h. Subsequently, dimethyl sulfoxide (DMSO) (160 µl/well) was added to dissolve the formazan, and the absorbance (OD) values were read using an microplate reader at 490 nm (Thermo Fisher Scientific, Inc.). The experiment was repeated three times.

Cell proliferation was tested using a BrdU kit (Roche Applied Science, Mannheim, Germany) on the basis of the instructions. Cells were plated in 96-well plates and transfections were performed. Cells were incubated with BrdU solution (10 µl/well) for 1.5 h and then denaturing solution (100 µl/well) for 25 min. Cells were then stained with anti-BrdU antibody for 1.5 h at room temperature followed by staining with secondary antibody solution. Next, 100 µl of tetramethyl benzidine substrate was added and allowed to incubate for 30 min. Finally, the results were detected at 450 nm using a SpectroFluor Plus multiwell plate reader (Tecan Group, Ltd., Mannedorf, Switzerland). The experiment was repeated three times.

Bio-Coat cell migration chambers (BD Biosciences, San Jose, CA, USA) were used to assess cervical cancer cell invasion. Chambers were coated with Matrigel (Becton Dickinson; BD Biosciences), and the transfected cells were suspended using 350 µl serum-free medium (1×10⁵ cells) and added to the top chamber. Then, the cells were cultured with 5% CO₂ at 37°C for 48 h followed by the addition of culture medium (500 µl) with 10% FBS to the lower chamber. Non-invading cells were gently removed using a cotton swab, and invasive cells were fixed with 95% ethanol and stained with trypan blue. The invasive cells were quantified as the mean count of stained cells in six random fields under bright field microscopy (×400 magnification) using an Olympus IX70 inverted microscope (Olympus Corp., Tokyo, Japan). The experiment was repeated three times.

Statistical significance was determined by the Student's t-test between two groups and by one-way ANOVA followed by a Bonferroni test for multiple groups. A P-value of <0.05 was considered to indicate a statistically significant result. Data are expressed as the mean ± standard deviation (SD). Statistical analyses were processed with SPSS version 22.0 software (IBM Corp., Armonk, NY, USA).

We quantified the expression of HOXA5 in cervical cancer cell lines and tissues to explore the expression of HOXA5 in cervical cancer. The results showed that mRNA expression (Fig. 1A) of HOXA5 as determined by RT-qPCR in cervical cancer cell lines and tissues was significantly decreased in both when compared with human cervical epithelial HCerEpic cells and normal tissues. The mRNA reduction of HOXA5 in ME-180, HT-3, HeLa and SiHa cells exhibited no significant differences between each cell line; thus we detected the protein expression of HOXA5 in ME-180 and HT-3 cells. The protein expression (Fig. 1B) of HOXA5 as determined by western blot analysis in ME-180 and HT-3 cells was also significantly decreased when compared with the HCerEPic cell line.

To investigate the potential role of HOXA5 in cervical cancer, we measured cell apoptosis by using Annexin V-FITC/PI assay and caspase-3 activity testing after HOXA5 overexpression in ME-180 and HT-3 cells. Firstly, we overexpressed HOXA5 by cells transfection with pcDNA.3.1-HOXA5, and the data indicated that mRNA (Fig. 2A) and protein (Fig. 2B) expression of HOXA5 were both increased twice. Thus, the gain-of-function experiment of HOXA5 was successful. Subsequently, the effect of HOXA5 upregulation on cell apoptosis in ME-180 and HT-3 cells were tested. In the Annexin V-FITC/PI assay (Fig. 2C), cell apoptosis was significantly induced by upregulation of HOXA5. Moreover, caspase-3 activity (Fig. 2D) was also significantly facilitated after pcDNA.3.1-HOXA5 transfection.

To further estimate the influence of HOXA5 overexpression on cervical cancer, we tested cell viability and proliferation using MTT and BrdU assays, respectively. The results revealed that cell viability (Fig. 3A) was markedly suppressed in the pcDNA.3.1-HOXA5 group when compared to that noted in the pcDNA.3.1group. We also assessed the proliferation (Fig. 3B) of cells transfected with pcDNA.3.1-HOXA5 for 24, 48 and 72 h, and it was showed that the proliferation in the pcDNA.3.1-HOXA5 (24 h) group was significantly inhibited in contrast to the pcDNA.3.1 group, and the suppression was slightly time-dependent. Additionally, cell invasion (Fig. 3C) was detected by Transwell invasion assay, and the data showed a significant decrease in cell invasion after HOXA5 overexpression. Therefore, HOXA5 displayed a valid inhibitory effect on cell proliferation and invasion in cervical cancer cell lines.

To gain insight into the molecular mechanisms of HOXA5 in cervical cancer, we determined the protein expression of p-AKT and p27 via western blot assay in ME-180 and HT-3 cells with HOXA5 upregulation. The results indicated that the protein level of p-AKT (Fig. 4) in the pcDNA.3.1-HOXA5 group was significantly reduced in contrast to the pcDNA.3.1 group. Meanwhile, p27 (Fig. 4) expression was increased in the pcDNA.3.1-HOXA5 group. Moreover, we treated ME-180 and HT-3 cells with SC79 (AKT activator, 0.1–10 µg/ml) for 1 h after HOXA5 overexpression. We found that SC79 (1–10 µg/ml) markedly activated AKT (p-AKT intensity increase) in both the ME-180 and HT-3 cells and the activity on p-AKT was dose-dependent (Fig. 5A). Thus, we measured the protein expression of p27 in ME-180 and HT-3 cells with HOXA5 overexpression and SC79 (1 µg/ml) incubation for 1 h, and the results showed that p27 was obviously downregulated (Fig. 5B). Meanwhile, caspase-3 activity (Fig. 5C) was inhibited and cell viability (Fig. 5D) and invasion (Fig. 5E) were elevated. Thus, HOXA5 controls cervical cancer cell proliferation and apoptosis via the regulation of p-AKT/p27.