Cervical cancer and paired adjacent normal tissues (n=30) were surgically collected from the Department of Obstetrics and Gynecology, Sanya People's Hospital (Sanya, China) from May 2018 to May 2020. The average age of all patients was 49 years (age range, 36–69 years). The clinical information of patients with cervical cancer is presented in Table SI. No patients received adjuvant treatment prior to surgery. In addition, the patients with other tumors or a history of treatments for other gynecological tumors were excluded from the present study. All tissue samples were immediately preserved in liquid nitrogen and stored at −80°C until RNA extraction. The present study was approved by the Ethics Committee of the Sanya People's Hospital (approval no. 2017.106; Sanya, China) and written informed consent was provided by all patients prior to the study start.

The Ect1/E6E7 normal human cervical epithelial cell line was purchased from the American Type Culture Collection, while the SiHa, HeLa, CaSki and ME180 human cervical cancer cell lines were purchased from the The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. All cells were maintained in DMEM (cat. no. SH30022.01B; Cytiva) supplemented with 10% fetal bovine serum (cat. no. 16140071; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (cat. no. 15140148; Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (cat. no. 15140122; Thermo Fisher Scientific, Inc.), at 37°C with 5% CO₂ and 100% humidity.

The overexpression vector pcDNA 3.1 was purchased from Thermo Fisher Scientific, Inc. All sequences were synthesized by Shanghai GenePharma Co., Ltd. The primers for the amplification of the full-length UCA1 were as follows: Forward, 5′-CCGGAATTCTGACATTCTTCTGGACAATG-3′ and reverse, 5′-CCGCTCGAGCTGACTCTTTTAGGAAGATTTCT-3′. The overexpression vector pcDNA-UCA1 was produced by cloning the full-length UCA1 into pcDNA3.1. For the knockdown of UCA1, the small interfering (si)RNAs targeting UCA1 (si-UCA1) were designed. The sequences was as follows: 5′-GGACAACAGUACACGCAUATT-3′. The sequences used to increase or decrease miR-299-3p expression were as follows: miR-299-3p mimics, 5′-UCGCCAAAUGGUAGGGUGUAU-3′; miR-299-3p inhibitor, 5′-AAGCGGUUUACCAUCCCACAU-3′; miR-NC mimics, 5′-UUCUCCGAACGUGUCACGUG-3′ and miR-NC inhibitor, 5′-CAGUACUUUUGUGUAGUACA-3′. The UCA1 overexpression vector pcDNA-UCA1, specific siRNAs targeting UCA1 (si-UCA1), negative control siRNA (si-NC), miR-299-3p mimics, NC mimics, miR-299-3p inhibitor and NC inhibitor were transfected into SiHa cells. For cell transfection, SiHa cells were seeded into a 6-well plate wat a density of 1×10⁵ cells/well. 1.5 ml of serum-free medium containing 500 µl of Lipofectamine™ 3000 transfection solution (cat. no. L3000015; Thermo Fisher Scientific, Inc.) were added in each well at 37°C for 48 h. After cell culture for 1–2 weeks, the positive colonies were harvested for amplification culture.

Total RNA was extracted from normal and tumor tissues and cell lines using TRIzol^® reagent (cat. no. 15596018; Thermo Fisher Scientific, Inc.). The reaction mixture containing 1 µg of total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent kit (cat. no. RR036A; Takara Bio, Inc.). The temperature protocols for RT were as follows: 50°C for 15 min and 85°C for 5 sec. The reactions were performed using an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the RNA expression levels were measured using a SYBR™ Green Master Mix (cat. no. RR430S; Takara Bio, Inc.). The thermocycling conditions of qPCR were as follows: Initial denaturation at 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 20 sec. The following primer sequences were used for qPCR: UCA1 forward, 5′-GCCAGCCTCAGCTTAATCCA-3′ and reverse, 5′-CCCTGTTGCTAAGCCGATGA-3′; miR-299-3p forward, 5′-ACACTCCAGCTGGGTATGTGGGATGGTAAAC-3′ and reverse, 5′-GTGCAGGGTCCGAG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′ reverse; and GAPDH forward, 5′-ACCCACATCCCTCAGACAC-3′ and reverse, 5′-CCCCAATACGACCAAATCC-3′. GAPDH and U6 were used as the internal controls. Relative expression levels were calculated using the 2^−ΔΔCq method (45). The experiments were performed in triplicate.

Cervical cancer cell proliferation was assessed via the CCK-8 assay (cat. no. C0039; Beyotime Institute of Biotechnology). The transfected cells were collected by centrifugation at 1,000 × g for 5 min at 4°C, then the transfected cells (2×10³ cells/100 µl) were cultured in 96-well plates. Following incubation for 0, 24, 48 and 72 h, 10 µl CCK-8 solution was added into each well and incubated for an additional 2 h at 37°C. Cell proliferation was subsequently analyzed at a wavelength of 450 nm. The experiments were performed in triplicate.

Cell invasion was assessed via the Transwell assay. The Transwell chambers were pre-coated with 100 µl Matrigel at 37°C for 1 h. Transfected cells were collected (1,000 × g for 5 min at 4°C), then the cells (1×10⁵ cells/well) were seeded into the upper chambers of Matrigel-coated Transwell plates (8-µm pore size; cat. no. 354483; Corning, Inc.) in serum-free DMEM. The DMEM (cat. no. SH30022.01B; Cytiva) medium with 10% fetal bovine serum (cat. no. 16140071; Thermo Fisher Scientific, Inc.) was plated in the lower chambers. Following incubation for 24 h at 37°C, cells in the lower chambers were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet dye (cat. no. 548-62-9; MilliporeSigma) for 30 min at room temperature, respectively. Stained cells were counted in five randomly selected fields using a BX63 microscope (magnification, ×100, Olympus Corporation).

LncBase Predicted v.2 software (46) was used to predict the potential binding sites between UCA1 and miR-299-3p.

The dual-luciferase reporter plasmids were constructed by inserting a UCA1 wild-type (UCA1-WT) or UCA1 mutant (UCA1-MUT) sequence into the psiCHECK-2 luciferase vector (Promega Corporation). SiHa cells were co-transfected with UCA1-WT or -MUT reporter and miR-299-3p or NC mimics, using Lipofectamine^® 3000 transfection reagent (cat. no. L3000015; Thermo Fisher Scientific, Inc.). Transfected cells were subsequently cultured in 24-well plates. Following incubation at 37°C for 24 h, luciferase activities were detected using a Dual-Luciferase Reporter assay system (cat. no. E1910; Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity (cat. no. E2810; Promega Corporation).

Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc.). All experiments were performed in triplicate and data are presented as the mean ± SD. Paired Student's t-test was used to compare differences between tumor tissues and adjacent normal tissues, while unpaired Student's t-test was used to compare differences between unpaired groups. One-way ANOVA followed by Tukey's or Dunnett's post hoc tests were used to compare differences between multiple groups. Pearson's correlation coefficient analysis was performed to assess the correlation between UCA1 and miR-299-3p expression. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis was performed to detect UCA1 expression in cervical cancer tissues and adjacent normal tissues (n=30). The results demonstrated that UCA1 expression was significantly upregulated in cervical cancer tissues compared with adjacent normal tissues (P<0.01; Fig. 1A). RT-qPCR analysis was also performed to detect UCA1 expression in the SiHa, HeLa, CaSki and ME180 cervical cancer cell lines and Ect1/E6E7 human normal cervical epithelial cell line. The results demonstrated that UCA1 expression was significantly upregulated in the cervical cancer cell lines compared with the normal cell line (all P<0.01; Fig. 1B). Taken together, these results suggest that UCA1 plays a key role in the development of cervical cancer.

To further investigate the effect of UCA1 in cervical cancer cell proliferation and invasion, SiHa cells were transfected with UCA1 or si-UCA1 to construct a UCA1 overexpression and knockdown model, respectively. The expression of UCA1 in transfected SiHa cells was detected via RT-qPCR analysis. The results demonstrated that UCA1 expression significantly increased (P<0.01) following transfection with UCA1 and significantly decreased (P<0.01) following transfection with si-UCA1 (Fig. 2A).

The CCK-8 assay was performed to assess the proliferation of cervical cancer cells. The results demonstrated that overexpression of UCA1 significantly promoted the proliferation of SiHa cells (P<0.01; Fig. 2B), the effects of which were reversed following UCA1 knockdown (P<0.01; Fig. 2C). Furthermore, the Transwell assay demonstrated that overexpression of UCA1 significantly promoted the invasive ability of SiHa cells (P<0.01; Fig. 2D), the effects of which were reversed following UCA1 knockdown. Collectively, these results suggest that UCA1 is involved in cervical cancer cell proliferation and invasion.

To clarify the molecular mechanism of UCA1 in cervical cancer, LncBase software was used to predict the binding sites between miRNAs and UCA1. The results revealed a potential binding site between UCA1 and miR-299-3p (Fig. 3A). The dual-luciferase reporter assay was subsequently performed to confirm the association between UCA1 and miR-299-3p in SiHa cells. The results demonstrated that transfection with miR-299-3p mimics significantly decreased (P<0.01) the luciferase activity of UCA1-WT, but not UCA1-MUT in SiHa cells (Fig. 3B). RT-qPCR analysis was performed to detect miR-299-3p expression in cervical cancer tissues and cell lines. The results demonstrated that miR-299-3p expression was significantly downregulated in cervical cancer tissues (P<0.01; Fig. 3C) and cell lines (all P<0.01; Fig. 3D). Pearson's correlation analysis was performed to determine the correlation between UCA1 and miR-299a-3p expression in cervical cancer tissues. The results demonstrated that miR-299-3p expression was negatively correlated with UCA1 expression (Fig. 3E). SiHa cells were transfected with pcDNA, UCA1, si-NC or si-UCA1, and miR-299-3p expression was detected. The results demonstrated that overexpression of UCA1 significantly decreased miR-299-3p expression (P<0.01), while UCA1 knockdown significantly increased miR-299-3p expression (P<0.01) in SiHa cells (Fig. 3F). Taken together, these results suggest that UCA1 binds to miR-299-3p and negatively regulates its expression in cervical cancer.

To determine the biological function of miR-299-3p in the proliferation and invasion of cervical cancer cells, SiHa cells were transfected with miR-299-3p mimics, NC mimics, miR-299-3p inhibitor or NC inhibitor. RT-qPCR analysis was performed to detect miR-299-3p expression. The results demonstrated that miR-229-3p expression significantly increased (P<0.01) following transfection with miR-299-3p mimics, the effects of which were reversed following transfection with miR-299-3p inhibitor (P<0.01), compared with the NC groups (Fig. 4A).

The proliferation and invasion of cervical cancer cells were assessed via the CCK-8 and Transwell assays, respectively. The results confirmed that overexpression of miR-299-3p significantly decreased cervical cancer cell proliferation (P<0.01; Fig. 4B), while miR-299-3p knockdown significantly increased cervical cancer cell proliferation (P<0.01; Fig. 4C). The Transwell assay demonstrated that transfection with miR-299-3p mimics significantly decreased cervical cancer cell invasion (P<0.01; Fig. 4D), while transfection with miR-299-3p inhibitor significantly promoted cervical cancer cell invasion (P<0.05; Fig. 4D). Collectively, these results suggest that miR-299-3p acts as a tumor suppressor in cervical cancer.

To further clarify the effect of the UCA1/miR-299-3p axis in cervical cancer tumorigenesis, rescue experiments were performed on SiHa cells. miR-299-3p mimics + UCA1 or miR-299-3p inhibitor + si-UCA1 were transfected into SiHa cells. RT-qPCR analysis demonstrated that overexpression of miR-299-3p partially reversed the inhibitory effects of overexpressing UCA1 on SiHa cells (P<0.05; Fig. 5A), while transfection with miR-299-3p inhibitor reversed the promoting effects of si-UCA1 on SiHa cells (P<0.01; Fig. 5B). Furthermore, transfection with miR-299-3p mimics inhibited the proliferative ability of UCA1-transfected SiHa cells (P<0.01; Fig. 5C), and si-UCA1-induced suppression in cell proliferation was recovered following transfection with miR-299-3p inhibitor (P<0.01; Fig. 5D). Furthermore, the invasive ability of UCA1-transfected SiHa cells decreased following transfection with miR-299-3p mimics (P<0.01; Fig. 5E), the effects of which were reversed following transfection with miR-299-3p inhibitor (P<0.05; Fig. 5F). Collectively, these results suggest that UCA1 promotes cervical cancer progression by regulating miR-299-3p expression.