Cervical squamous cell carcinoma cell lines (Ca Ski and SiHa), a endocervical adenocarcinoma cell line (HeLa) and the normal cervical epithelial cell line (End1/E6E7) were obtained from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. All cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/l and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/l penicillin (Gibco; Thermo Fisher Scientific, Inc.) and maintained in an atmosphere of 37°C with 5% CO₂.

pIRES vectors containing short hairpin RNA (shRNA)-circ-ACACA#1, shRNA-circ-ACACA#2, shRNA-negative control (NC), miR-582-5p mimic (50 nM; sense, 5′-UUACAGUUCAACCAGUUACU-3′ and antisense, 5′-UAACUGGUUGAACAACUGUAAUU-3′), mimic-NC (50 nM; sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′), miR-582-5p inhibitor (50 nM; 5′-AGUAACUGGUUGAACAACUGUAA-3′), NC inhibitor (50 nM; 5′-CAGUACUUUUGUGUAGUACAAA-3′), pcDNA3.1-ERO1A and pcDNA3.1-NC (empty plasmid) were purchased from Shanghai GenePharma Co., Ltd. HeLa cells (1×10⁶ cells per well) were plated and incubated in six-well plates for 24 h and then transfected for 48 h with the corresponding plasmids, oligonucleotides or NCs using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. The transfected cells were used for subsequent experiments at 48 h after transfection. Untreated cells were used as the control group.

Total RNA was extracted from CC cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers protocols. Total RNA was reverse transcribed into cDNA using the PrimeScript™ Strand cDNA synthesis kit (Takara Biotechnology Co., Ltd.). The temperature protocol for this step was as follows: 70°C for 5 min, 37°C for 5 min and 42°C for 1 h. qPCR was subsequently performed using a Power SYBR^® Green PCR Master mix (Invitrogen; Thermo Fisher Scientific, Inc.) on an ABI 7500 Real-Time PCR Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: Initial denaturation at 95°C for 10 min; followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 1 min; and a final extension of 10 min at 72°C. Primers pairs used in this study were as follows: circ-ACACA forward, 5′-GTGGCTTTGAAGGAGCTGTC-3′ and reverse, 5′-CAGACATGCTGGACCTTGAA-3′; miR-582-5p forward, 5′-GCGGTTACAGTTGTTCAACC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; ERO1A forward, 5′-ATGACATCAGCCAGTGTGGA-3′ and reverse, 5′-CATGCTTGGTCCACTGAAGA-3′; GAPDH forward, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and reverse, 5′-GCCATCACGCCACAGTTTC-3′; and U6 forward, 5′-GGAACGATACAGAGAAGATTAGC-3′ and reverse, 5′-TGGAACGCTTCACGAATTTGCG-3′. The miRNA expression level was normalized to U6 and GAPDH was applied as the internal control of mRNAs, following quantification using the 2^−∆∆Cq method (23).

Six units of RNase R (Geneseed Biotech, Inc.) were added into every 2 µg RNA and incubated at 37°C for 20 min. After RNase R treatment, the mRNA expression of circ-ACACA and ACACA was detected by RT-qPCR.

Cell cytoplasm/nucleus fraction isolation was performed using the Nuclear/Cytosol Fractionation Kit (Cell Biolabs, Inc.). Briefly, extracted RNAs from the cytoplasm or nucleus were determined by RT-qPCR which was performed in the previous RT-qPCR section. The relative expression levels of circ-ACACA, ACACA, nuclear control transcript (U6) and cytoplasmic control transcript (β-actin) were measured. β-actin and U6 were used as the internal control for cytosolic and nuclear fractions, respectively.

The viability of transfected HeLa cells was detected using MTT reagent (Sigma-Aldrich; Merck KGaA). Briefly, cells (6×10³ cells/well) were plated into 96-well plates and incubated for 24, 48 or 72 h. At each time point, 10 µl MTT was added to each well. Following incubation, 100 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to each well to dissolve the purple formazan crystals. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

A total of 2×10³ HeLa cells/well were seeded into a six-well plate and maintained in a humidified incubator with 5% CO₂ at 37°C. Following 48 h of incubation, the plates were washed with PBS twice, and then colonies were fixed with 10% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. After the plates were washed with phosphate buffer saline (PBS) at room temperature for 1 min and dried, the number of colonies was counted under an inverted light microscope.

To detect the ability of cell invasion, Transwell plates with a 8-µm pore insert precoated with Matrigel (BD Biosciences) overnight at 37°C were obtained. A total of 2×10⁴ transfected HeLa cells were trypsinized and plated into the upper chamber of transwell plates with serum-free DMEM. The lower chambers were filled with 800 µl DMEM supplemented with 10% FBS. Following 48 h of incubation at 37°C, the invasive cells were fixed with 95% ethanol for 20 min at 37°C and stained with 0.1% crystal violet for 10 min at 37°C. The number of cells in five randomly selected fields of view was counted under a light microscope.

HeLa cells were plated into 96-well plates at a density of 4×10³ cells/well and cultured until they reached 80% confluence. The cells were washed with PBS three times and then cultured in medium supplemented with 1% FBS for 24 h. An artificial wound was created across the cell monolayer by scratching the center of all wells with a 10-µl pipette tip. Migratory cells were visualized using an inverted light microscope. ImageJ software (version 1.52r; National Institutes of Health) was used to determine the wound healing rate.

Following culture for 24 h, 1×10⁵ HeLa cells were seeded in a 6-well plate. Cells were fixed with 4% paraformaldehyde at 4°C for 20 min, permeabilized with 0.1% Triton X-100 in PBS and subsequently incubated with TUNEL reagents (EMD Millipore). Cell nuclei was stained with diaminobenzene for 10 min at room temperature. The apoptosis of TUNEL-positive cells was observed under a fluorescence microscope (magnification, ×200; Olympus Corporation) and cells were counted in five randomly selected microscopic fields.

Glycolysis was determined using a Seahorse XF Glycolytic Rate assay kit (Agilent Technologies, Inc.) on a Seahorse XFe96 analyzer (Agilent Technologies, Inc.) according to the manufacturers protocol. Briefly, 2×10⁴ HeLa cells per well transfected with shRNA-circ-ACACA, miR-582-5p, shRNA-circ-ACACA + miR-582-5p inhibitor or respective NCs were plated into a 96-well plate. After the probes were calibrated, 10 mmol glucose, 10 µmol oligomycin and 50 mmol 2-deoxyglucose were serially injected to detect the extracellular acidification rate (ECAR). Data were analyzed using Seahorse XFe24 Wave 2.2 software (Agilent Technologies, Inc.).

HeLa cells and cell culture medium were collected following transfection for 48 h. The concentration of glucose in the cell culture medium was determined using a Glucose assay kit (Sigma-Aldrich; Merck KGaA), whereas the lactate concentration was measured using a Lactic Acid assay kit (Seebio), according to the manufacturers protocols.

¹³C-labeled intracellular metabolites were identified as previously described (24). Briefly, 1×10⁷ HeLa cells were incubated with 2 g/l ¹³C-labeled glucose for 2 h at 37°C. Metabolites were subsequently extracted and evaluated using a liquid chromatography system equipped with a TripleTOF^® 5600 mass spectrometer (SCIEX; AB SCIEX). Electrospray positive ion mode was used along with the following parameters: ion voltage, 5,500 V; declustering potential, 80 V; source temperature, 60°C; curtain gas, 35 psi; and 100–1,000 m/z.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology) and protein samples (40 µg protein/lane) were separated via 12% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes (EMD Millipore) and the membranes were blocked with 5% skimmed milk for 1.5 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C. Following the primary antibody incubation, the membranes were rinsed with TBS-0.2% Tween 20 and incubated with the goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (dilution, 1:2,000; cat. no. ab205718; Abcam) at room temperature for 1 h. GAPDH was used as the internal loading control. Protein bands were visualized using an ECL detection system (Thermo Fisher Scientific, Inc.) and densitometric analysis was performed using ImageJ software (version 1.52r; National Institutes of Health). The following primary antibodies were used: anti-hypoxia-inducible factor 1α (HIF-1A; cat. no. 36169T; 1:1,000; Cell Signaling Technology, Inc.), anti-lactate dehydrogenase A (LDHA; cat. no. 3582T; 1:1,000; Cell Signaling Technology, Inc.), anti-glucose transporter type 1 (GLUT1; cat. no. 12939S; 1:1,000; Cell Signaling Technology, Inc.), anti-hexokinase-2 (HK2; cat. no. 2867S; 1:1,000; Cell Signaling Technology, Inc.), anti-ERO1A (cat. no. 3264T; 1:1,000; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. 5174T; 1:1,000; Cell Signaling Technology, Inc.). GAPDH was used as an internal control.

The binding site between circ-ACACA and miR-582-5p as well as miR-582-5p and ERO1A was predicted using the Encyclopedia of RNA Interactomes (ENCORI) database (http://starbase.sysu.edu.cn/). To validate the binding relationship between miR-582-5p and circ-ACACA or ERO1A, wild-type (WT) or mutant (MUT) 3-untranslated region sequences of circ-ACACA or ERO1A were cloned into a pmiRGLO dual-luciferase miRNA target expression reporter (Promega Corporation). HeLa cells were subsequently transfected with miR-582-5p mimic or mimic-NC and WT or MUT reporters using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The relative luciferase activity was measured at 48 h post-transfection using a Dual Luciferase Reporter assay system (Promega Corporation). And firefly luciferase activity was normalized to Renilla luciferase activity.

All experiments were repeated independently in triplicate. Statistical analysis was performed using SPSS v20.0 software (IBM Corp.). Data are presented as the mean ± SD. Statistical differences between two groups were determined using an unpaired Students t-test, while one-way ANOVA followed by Tukeys post hoc test was used for comparisons among ≥3 groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of circ-ACACA in CC, its expression levels in CC cells were first determined using RT-qPCR. The results revealed that the mRNA expression levels of circ-ACACA were upregulated in CC cells compared with End1/E6E7 cells (Fig. 1A). The expression levels of circ-ACACA were the highest in HeLa cells among the cell lines examined. Therefore, HeLa cells were used as the CC cell model in subsequent experiments. As shown in Fig. 1B, circ-ACACA was not dissolved, while ACACA mRNA was dissolved by RNase R. Furthermore, circ-ACACA was expressed at a higher level in the cytoplasm compared with the nucleus (Fig. 1C). These results suggested that circ-ACACA expression may be upregulated in CC cells.

circ-ACACA expression was subsequently knocked down to determine its effects in CC cells. The expression levels of circ-ACACA were downregulated following transfection with shRNA-circ-ACACA#1 and shRNA-circ-ACACA#2 compared with shRNA-NC, especially in shRNA-circ-ACACA#2. Therefore, shRNA-circ-ACACA#2 was selected for use in subsequent experiments (Fig. 2A). The effects of transfection of shRNA-circ-ACACA on CC cells were determined using functional assays. MTT and colony formation assays demonstrated that cell viability and proliferation were decreased in HeLa cells transfected with shRNA-circ-ACACA as compared with the shRNA-NC group (Fig. 2B and C). Furthermore, transfection with shRNA-circ-ACACA increased the levels of apoptosis compared with the shRNA-NC group (Fig. 2D and E). circ-ACACA silencing also notably inhibited cell invasion and migration compared with the shRNA-NC group (Fig. 2F-I). These results suggested that silencing of circ-ACACA may inhibit the proliferation, invasion and migration, and promote the apoptosis of CC cells.

It was subsequently investigated whether knockdown of circ-ACACA led to the attenuation of glycolysis. As displayed in Fig. 3A, ECAR was notably decreased after transfection with shRNA-circ-ACACA when compared with the shRNA-NC group. Lactate production and glucose consumption assays were performed to evaluate the effect of circ-ACACA on glycolysis. As shown in Fig. 3B and C, the transfection with shRNA-circ-ACACA markedly decreased the production of lactate and increased the levels of glucose compared with the shRNA-NC group. In addition, HeLa cells were incubated with ¹³C-labeled glucose and liquid chromatography-mass spectrometry was performed to gain more in-depth insights into the metabolic flux of glucose. As shown in Fig. 3D, the ratio of 13C-marked glucose was notably decreased following circ-ACACA-knockdown relative to the shRNA-NC group. As expected, the expression levels of glycolysis-related proteins, including HIF-1A, LDHA, GLUT1 and HK2, were markedly downregulated following the knockdown of circ-ACACA compared with those in the shRNA-NC group (Fig. 3E). These results indicated that silencing of circ-ACACA may inhibit glycolysis in CC cells.

To determine the regulatory effect of circ-ACACA on the expression levels of miR-582-5p, the expression levels of miR-582-5p were first analyzed in various CC cell lines. The results revealed that miR-582-5p expression was downregulated in CC cells compared with End1/E6E7 cells (Fig. 4A). Subsequently, a binding site between circ-ACACA and miR-582-5p was predicted using ENCORI (Fig. 4B). Following the overexpression of miR-582-5p by transfection with the miR-582-5p mimic, a dual-luciferase reporter assay was used to demonstrate the binding relationship between circ-ACACA and miR-582-5p (Fig. 4C and D). As shown in Fig. 4E, knockdown of circ-ACACA markedly upregulated the expression levels of miR-582-5p compared with those in the shRNA-NC group. These data suggested that circ-ACACA may negatively regulate miR-582-5p expression in HeLa cells.

ERO1AIt was hypothesized that circ-ACACA may exert its promoting effects on the progression of CC via the miR-582-5p/ERO1A signaling axis. To verify this hypothesis, the expression levels of ERO1A in various CC cell lines were determined. As illustrated in Fig. 5A and B, ERO1A expression was notably unregulated in CC cells compared with End1/E6E7 cells. Since ERO1A was predicted to target and bind with miR-582-5p using ENCORI database. A binding association between ERO1A and miR-582-5p was predicted using ENCORI (Fig. 5C) and verified using a dual-luciferase reporter assay (Fig. 5D). Subsequently, RT-qPCR and western blotting demonstrated that transfection with the miR-582-5p mimic markedly reduced the mRNA and protein expression levels of ERO1A (Fig. 5E and F). Furthermore, transfection with shRNA-circ-ACACA markedly downregulated the mRNA and protein expression levels of ERO1A compared with the shRNA-NC group (Fig. 5G and H). These findings suggested that ERO1A may be a target of miR-582-5p, and that circ-ACACA may upregulate the expression levels of ERO1A by sponging and negatively regulating miR-582-5p expression.

To investigate whether circ-ACACA could affect the functions of CC cells in vitro via the miR-582-5p/ERO1A signaling axis, ERO1A was overexpressed in CC cells. The expression levels of ERO1A were markedly upregulated in the pcDNA3.1-ERO1A group compared with the pcDNA3.1-NC group, demonstrating the successful establishment of ERO1A-overexpressing CC cells (Fig. 6A and B). Additionally, miR-582-5p expression was notably downregulated after transfection with miR-582-5p inhibitor compared with that in the NC inhibitor group (Fig. 6C). Knockdown of circ-ACACA reduced the viability, proliferation, invasion and migration, and enhanced the apoptosis of HeLa cells, and these effects were all partially reversed following the transfection with the miR-582-5p inhibitor or pcDNA3.1-ERO1A (Fig. 6D-K). In addition, transfection with circ-ACACA reduced the levels of ECAR, and these effects were subsequently reversed by transfection with the miR-582-5p inhibitor or pcDNA3.1-ERO1A (Fig. 7A). Furthermore, the results of the lactate production and glucose consumption assays revealed that the decreased lactate production and enhanced glucose levels induced by transfection with shRNA-circ-ACACA were partially recovered following the transfection with the miR-582-5p inhibitor or pcDNA3.1-ERO1A (Fig. 7B and C). Finally, the shRNA-circ-ACACA-induced decreases in the ratio of ¹³C-marked glucose and expression levels of glycolysis-related proteins were partially reversed following transfection with the miR-582-5p inhibitor or pcDNA3.1-ERO1A (Fig. 7D and E). Overall, these results suggested that knockdown of miR-582-5p or overexpression of ERO1A alleviates the effects of circ-ACACA silencing on CC cell proliferation, viability, invasion, migration and apoptosis.