In total, 49 pairs of cervical cancer tissues and adjacent non-cancerous tissues were collected from patients (age range, 46–72 years; mean age, 63 years) who underwent surgical resection at Huzhou Central Hospital (Zhejiang, China) between May 2014 and March 2016. None of the patients had been treated with chemotherapy or radiotherapy prior to surgery. All fresh tissues were quickly frozen in liquid nitrogen and stored at −80°C. The present study was approved by the Ethics Committee of Huzhou Central Hospital, and written informed consent was provided by all participants.

A total of four cervical cancer cell lines (SiHa, HeLa, C-33A and CaSki) and a normal human cervix epithelial cell line (Ect1/E6E7) were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% v/v fetal bovine serum (FBS), 1% v/v penicillin/streptomycin mixture (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and maintained at 37°C in an incubator with 5% CO₂ and 95% air.

miR-874 mimics, negative control miRNA mimics (miR-NC), small interfering RNA (siRNA) targeting the expression of ETS1 (ETS1 siRNA) and negative control siRNA (NC siRNA) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences used were as follows: miR-874 mimic, 5′-CUGCCCUGGCCCGAGGGACCGA-3′; miR-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; ETS1 siRNA, 5′-ACUUGCUACCAUCCCGUACTT-3′; and NC siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. In order to restore ETS1 expression, the ETS1 overexpression plasmid pCMV-ETS1 and empty plasmid pCMV, which were chemically synthesised by the Chinese Academy of Sciences (Shanghai, China), were applied. For cell transfection, cells were inoculated into 6-well plates one day prior to transfection. miRNA mimics (100 pmol), siRNA (100 pmol) or plasmids (4 µg) were transfected into cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. At 8 h post-transfection, the cell culture medium was discarded, and fresh DMEM containing 10% v/v FBS was added into each well.

Total RNA from tissue specimens and culture cells was isolated using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. To quantify miR-874 expression, reverse transcription was performed using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The temperature protocol for reverse transcription was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. Next, a TaqMan miRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was adopted for qPCR, with U6 small nuclear RNA as an internal reference. The thermocycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec; and annealing/extension at 60°C for 60 sec. To determine the level of ETS1 mRNA, complementary DNA (cDNA) was produced from total RNA using a PrimeScript RT reagent kit and this DNA was then subjected to qPCR using a SYBR Premix Ex Taq™ (both Takara Biotechnology Co., Ltd., Dalian, China). The temperature protocol for reverse transcription was as follows: 37°C for 15 min and 85°C for 5 sec. qPCR was performed using the following thermocycling conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. The primers were designed as follows: miR-874 forward, 5′-TGCGGCTGCCCTGGCCCGAGGGAC-3′ and reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; ETS1 forward, 5′-CCACCACTACTACCGAAA-3′ and reverse, 5′-AACACTTCTGCTTGATGGC-3′; and GAPDH forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. GAPDH served as an internal control for ETS1 mRNA expression. The relative expression of miR-874 and ETS1 was analysed using the 2^−ΔΔCq method (22).

CCK-8 assay was performed to determine cell proliferative ability. In brief, transfected cells were seeded into 96-well plates at a density of 3×10³ cells per well and incubated at 37°C under 5% CO₂ for 0, 1, 2 or 3 days. At indicated time points, CCK-8 assay was initiated by adding 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) into each well. Following incubation at 37°C for 2 h, the absorbance value of each well was detected at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

At 24 h after transfection, cells were harvested and prepared into a single-cell suspension. Transfected cells were plated into 6-well plates at a density of 1,000 cells/well and were then incubated at 37°C in an incubator under 5% CO₂ and 95% air for 14 days. At day 15, the cells were fixed with 95% methanol for 20 min at room temperature and stained with methyl violet (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 20 min. The number of cell colonies (>50 cells/colony) was counted under an inverted microscope (×200 magnification; IX83; Olympus Corporation, Tokyo, Japan).

Transfected cells were incubated at 37°C in an incubator under 5% CO₂ and 95% air for 48 h. Subsequently, the transfected cells were collected, washed with PBS and then subjected to cell apoptosis detection using an Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (BioLegend, Inc., San Diego, CA, USA). In brief, transfected cells were resuspended with 100 µl binding buffer. Next, the transfected cells were stained with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) at room temperature. Following incubation for 20 min in the dark, the cell apoptosis rate was detected using a flow cytometer (FACScan; BD Biosciences, Franklin Lakes, NJ, USA), and analyzed using CellQuest software version 3.3 (BD Biosciences, Franklin Lakes, NJ, USA).

Cell migration and invasion abilities were determined using Transwell chambers (Corning Incorporated, Corning, NY, USA) coated without or with Matrigel (BD Biosciences), respectively. Transfected cells were collected at 48 h post-transfection and were resuspended with FBS-free DMEM. The upper Transwell chambers were filled with 1×10⁵ cells in FBS-free DMEM, and the lower chambers were filled with 500 µl DMEM containing 10% FBS. The chambers were incubated at 37°C in an atmosphere of 5% CO₂ and 95% air for 24 h. The cells were fixed with 95% methanol at room temperature for 15 min, stained with 0.5% crystal violet at room temperature for 15 min and washed with PBS. The number of migrated and invaded cells in five randomly selected visual fields/chambers was counted under an inverted microscope at ×200 magnification.

TargetScan7.1 (http://www.targetscan.org/) and microRNA.org (http://www.microrna.org/microrna/home.do) were applied to predict the putative targets of miR-874. ETS1 was predicted as a major potential target of miR-874. The wild-type (Wt) and mutant (Mut) 3′-UTR of ETS1 was produced by Shanghai GenePharma Co., Ltd. and inserted into the pGL3 luciferase vector (Promega Corporation, Madison, WI, USA) to generate pGL3-ETS1-3′-UTR Wt and pGL3-ETS1-3′-UTR Mut, respectively. Cells were inoculated into 24-well plates at a density of 1.0×10⁵ cells each well. Following overnight incubation, miR-874 mimics or miR-NC, in combination with pGL3-ETS1-3′-UTR Wt or pGL3-ETS1-3′-UTR Mut, were transfected into cells using Lipofectamine 2000 reagent. Luciferase activities were detected at 48 h post-transfection using a Dual Luciferase Reporter assay kit (Promega Corporation), according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity.

Total protein was extracted from tissue specimens or culture cells using radioimmunoprecipitation assay buffer, and the concentration of total protein was quantified using a bicinchoninic acid assay kit (both Beyotime Institute of Biotechnology, Inc., Shanghai, China). Subsequently, 10% SDS-PAGE was utilised to separate equal amounts of proteins (20 µg); the separated proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, membranes were blocked with 5% skimmed milk that was dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with monoclonal anti-mouse ETS1 antibody (1:1,000 dilution; cat. no. ab96478; Abcam, Cambridge, UK) and monoclonal anti-rabbit GAPDH (1:1,000 dilution; cat. no. ab9484; Abcam) primary antibodies overnight at 4°C. Following washing three times with TBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; cat. no. ab6789; Abcam) at room temperature for 2 h. The protein signals were visualised using an enhanced chemiluminescence plus reagent (GE Healthcare, Chicago, IL, USA). Protein expression was quantified using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis was performed using SPSS software (version 18.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard deviation from ≥3 independent experiments and analysed using Student's t-test or one-way analysis of variance (ANOVA). Student-Newman-Keuls test was applied for post hoc analysis following ANOVA. The association between miR-874 and the clinicopathological features of patients with cervical cancer was analysed using χ² test. Spearman's correlation analysis was performed to evaluate the correlation between miR-874 and ETS1 mRNA in cervical cancer tissues. P<0.05 were considered to indicate a statistically significant difference.

To clarify the expression pattern of miR-874 in cervical cancer, miR-874 expression was initially detected in 49 pairs of cervical cancer tissues and adjacent non-cancerous tissues. The results of RT-qPCR analysis demonstrated that miR-874 expression was downregulated in cervical cancer tissues compared with that in adjacent non-cancerous tissues (P<0.05; Fig. 1A). To determine the clinical significance of miR-874 in cervical cancer, the association between miR-874 and the clinicopathological features of patients with cervical cancer was determined. All patients were divided into either low- or high-expression groups based on the median expression of miR-874. The results demonstrated that reduced miR-874 levels were correlated with the FIGO stage (P=0.007) and lymph node metastasis (P=0.032). However, no significant correlation was observed with other clinicopathological factors, including age, tumour size or family history of cancer (all P>0.05; Table I). The expression level of miR-874 was also determined in four cervical cancer cell lines (SiHa, HeLa, C-33A and CaSki) and a normal human cervix epithelial cell line (Ect1/E6E7). miR-874 expression was downregulated in all the tested cervical cancer cell lines, compared with that in Ect1/E6E7 cells (P<0.05; Fig. 1B). These results suggested that miR-874 is associated with cervical cancer progression.

To illustrate the biological roles of miR-874 in cervical cancer, the SiHa and HeLa cells were used, which exhibited relatively lower miR-874 expression compared with the two other cervical cancer cell lines in subsequent functional assays. SiHa and HeLa cells were transfected with miR-874 mimics or miR-NC. The results of RT-qPCR analysis confirmed that miR-874 was markedly overexpressed in SiHa and HeLa cells following transfection with miR-874 mimics (P<0.05; Fig. 2A). CCK-8 assay was performed to detect the proliferation of SiHa and HeLa cells that were transfected with miR-874 mimics or miR-NC. Ectopic miR-874 expression significantly decreased the proliferative abilities of SiHa and HeLa cells (P<0.05; Fig. 2B). To confirm this observation, a colony formation assay was used to evaluate the effect of miR-874 overexpression on colony formation ability in cervical cancer. Similarly, miR-874 upregulation significantly reduced the number and size of surviving colonies compared with the miR-NC groups (Fig. 2C; P<0.05). Flow cytometric assay was employed to examine the biological functions of miR-874 in cervical cancer cell apoptosis. The results revealed that resumption of miR-874 expression increased the apoptotic rate of SiHa and HeLa cells (Fig. 2D; P<0.05). Taken together, these results suggested that miR-874 inhibits cell proliferation and promotes cell apoptosis in cervical cancer.

Considering the association between miR-874 and lymph node metastasis, Transwell migration and invasion assays were utilised to investigate the effect of miR-874 overexpression on cervical cancer cell metastasis. SiHa and HeLa cells transfected with miR-874 mimics exhibited significantly lower migration (P<0.05; Fig. 3A) and invasion (P<0.05; Fig. 3B) abilities, compared with those transfected with miR-NC. These results suggested that miR-874 serves a tumour-suppressive role in cervical cancer metastasis.

The biological roles of miRNA depend on its specific target. Therefore, bioinformatic analysis was performed to predict the putative target genes of miR-874. According to TargetScan7.1 and microRNA.org, ETS1 was a predicted target gene of miR-874 (Fig. 4A). Luciferase reporter assay was conducted to investigate whether miR-874 directly targets the 3′-UTR of ETS1 in cervical cancer cells. miR-874 overexpression significantly suppressed the luciferase activities of the plasmid carrying the Wt binding site in SiHa and HeLa cells (P<0.05). However, the upregulation of miR-874 did not serve an inhibitory role in the luciferase activities of the plasmid harbouring the Mut binding site (Fig. 4B).

To further evaluate the association between miR-874 and ETS1 in cervical cancer, ETS1 expression was detected in 49 pairs of cervical cancer tissues and adjacent non-cancerous tissues through RT-qPCR. Results revealed that the levels of ETS1 mRNA were significantly higher in the cervical cancer tissues than in the adjacent non-cancerous tissues (P<0.05; Fig. 4C). The ETS1 mRNA levels were also identified to be inversely correlated with miR-874 expression in cervical cancer tissues (r=−0.5419; P<0.0001; Fig. 4D). The effects of miR-874 overexpression on ETS1 mRNA and protein levels in SiHa and HeLa cells were illustrated by RT-qPCR and western blot analysis, respectively. Restoration of miR-874 expression decreased ETS1 expression in SiHa and HeLa cells at mRNA (P<0.05; Fig. 4E) and protein (P<0.05; Fig. 4F) levels. Taken together, these results indicated that ETS1 is a direct target gene of miR-874 in cervical cancer.

Considering that ETS1 is a direct target gene of miR-874 in cervical cancer, we hypothesised that inhibiting ETS1 can mimic the biological functions of miR-874 in cervical cancer cells. SiHa and HeLa cells were transfected with ETS1 siRNA to knock down endogenous ETS1 expression. Following transfection, western blot analysis revealed that ETS1 protein expression was silenced effectively in the SiHa and HeLa cells transfected with ETS1 siRNA (P<0.05; Fig. 5A). As expected, inhibition of ETS1 significantly reduced the proliferative (P<0.05; Fig. 5B) and colony-forming abilities (P<0.05; Fig. 5C) of SiHa and HeLa cells. ETS1-knockdown also promoted the apoptosis (P<0.05; Fig. 5D) and prohibited the metastasis (P<0.05; Fig. 5E and F) of SiHa and HeLa cells. These results further demonstrated that ETS1 is a direct target gene of miR-874 in cervical cancer cells.

To investigate whether ETS1 is a functional mediator of miR-874, rescue experiments were performed by transfecting pCMV-ETS1 or pCMV into miR-874-overexpressing SiHa and HeLa cells. Following transfection, ETS1 protein levels were detected using western blot analysis. The decrease in ETS1 protein level induced by miR-874 overexpression was significantly restored in the SiHa and HeLa cells following co-transfection with pCMV-ETS1 (P<0.05; Fig. 6A). Furthermore, recovered ETS1 expression counteracted the effects of miR-874 overexpression on the proliferation (P<0.05; Fig. 6B), colony formation (P<0.05; Fig. 6C), apoptosis (P<0.05; Fig. 6D), migration (P<0.05; Fig. 6E) and invasion (P<0.05; Fig. 6F) of SiHa and HeLa cells. These findings clearly demonstrated that miR-874 may serve as a tumour suppressor in cervical cancer progression, at least partly, by suppressing ETS1 expression.