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Introduction

Cervical cancer is the third most common type of gynaecological cancer and the fourth leading cause of cancer-related mortality worldwide (1). A total of 530,000 new cases and 275,000 mortalities were estimated to be caused by cervical cancer annually worldwide (2). Currently, the mainstay therapy for patients with cervical cancer includes surgical resection, radiotherapy, chemotherapy and other comprehensive treatments (3). Despite substantial advances in diagnostic technologies and therapeutic methods, the therapeutic outcomes of patients with cervical cancer remain unsatisfactory, particularly for those diagnosed with an advanced stage of disease (4). Distant metastasis, lymph node recurrence and cancer recurrence are primarily responsible for the unfavourable prognosis of patients with cervical cancer (5). Persistent human papillomavirus (HPV) infection has been identified as a primary cause of cervical cancer (6). However, the detailed mechanisms of the pathogenesis of cervical cancer remain largely elusive. Therefore, further investigation into the mechanisms underlying the tumour onset and aggressiveness of cervical cancer is required in order to identify novel therapeutic targets that may improve the prognosis of patients with this disease.

MicroRNAs (miRNAs) are a large group of endogenous, single-stranded, non-coding RNA molecules comprising 18–25 nucleotides (7). miRNAs regulate genes expression by directly interacting with the complementary sites from the 3′-untranslated regions (3′-UTRs) of their target genes, causing mRNA degradation and/or translation inhibition (8). One miRNA can modulate numerous target genes; therefore, miRNAs naturally serve as key regulators of various physiological and pathological behaviours, including cell proliferation, cycle, apoptosis, differentiation, metabolism and metastasis (9). Recent studies have revealed that miRNAs are deregulated in the majority of human malignancies and that their deregulation is involved in tumourigenesis and tumour development (10,11). In cervical cancer, various miRNAs are aberrantly expressed, including miR-379 (12), miR-383 (13), miR-466 (14) and miR-1297 (15). Furthermore, miRNAs may serve tumour-suppressing or oncogenic roles in cervical cancer, depending on the detailed roles of their target genes (16,17). Therefore, miRNAs may be identified as promising therapeutic targets for treating patients with cervical cancer.

Recent studies have reported that miR-874 is frequently dysregulated in multiple types of human cancer, and that it acts as a key player in tumourigenesis and tumour development (18–21). However, the expression pattern, impacts and underlying mechanisms of miR-874 in cervical cancer have not been elucidated. Therefore, the present study measured miR-874 expression, analysed the biological roles of miR-874 and investigated its molecular mechanisms in cervical cancer cells.

Materials and methods

Tissue samples

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.

Cell culture

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.

Transfection assay

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.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

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).

Cell Counting kit-8 (CCK-8) assay

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).

Colony formation assay

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).

Flow cytometric assay

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).

Transwell migration and invasion assays

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.

Bioinformatics analysis and luciferase reporter assay

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.

Western blot analysis

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

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.

Results

miR-874 is downregulated in cervical cancer tissues and cell lines

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.

Figure 1. miR-874 expression is reduced in cervical cancer tissues and cell lines. (A) The expression level of miR-874 was determined by RT-qPCR in 49 pairs of cervical cancer tissues and adjacent non-cancerous tissues. *P<0.05 vs. non-cancerous tissues. (B) RT-qPCR analysis was performed to examine the expression of miR-874 in four cervical cancer cell lines (SiHa, HeLa, C-33A and CaSki) and a normal human cervix epithelial cell line (Ect1/E6E7). *P<0.05 vs. Ect1/E6E7. miR, microRNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Table I. Association between miR-874 and clinicopathological features of patients with cervical cancer.

Table I.

Association between miR-874 and clinicopathological features of patients with cervical cancer.

	miR-874 expression
Clinicopathological features	Low	High	P-value
Age, years			0.478
<60	7	9
≥60	18	15
Tumour size, cm			0.674
<4	11	12
≥4	14	12
Family history of cancer			0.291
Yes	5	8
No	20	16
FIGO stage			0.007
I–II	7	16
III–IV	18	8
Lymph node metastasis			0.032
No	7	14
Yes	18	10

[i] miR, microRNA; FIGO, International Federation of Gynecology and Obstetrics.

miR-874 inhibits the proliferation and promotes the apoptosis of cervical cancer cells

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.

Figure 2. Upregulation of miR-874 inhibits the proliferation and induces the apoptosis of SiHa and HeLa cells. (A) SiHa and HeLa cells were transfected with miR-874 mimics or miR-NC, and reverse transcription-quantitative polymerase chain reaction was utilised to detect the expression level of miRNA. (B) Cell Counting kit-8 assay was used to evaluate the effect of miR-874 overexpression on SiHa and HeLa cell proliferation. (C) Colony formation assay was performed to detect the colony formation ability of SiHa and HeLa cells transfected with miR-874 mimics or miR-NC. (D) Flow cytometric analysis was conducted to detect the apoptosis rates of SiHa and HeLa cells transfected with miR-874 mimics or miR-NC. *P<0.05 vs. miR-NC. miR, microRNA; NC, negative control.

miR-874 restricts cell migration and invasion 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.

Figure 3. miR-874 overexpression decreases the migration and invasion abilities of SiHa and HeLa cells. (A) Transwell migration assay was conducted to determine the migration ability of SiHa and HeLa cells following transfection with miR-874 mimics or miR-NC. (B) The effect of miR-874 overexpression on the invasion capacity of SiHa and HeLa cells was determined using Transwell invasion assay. *P<0.05 vs. miR-NC. miR, microRNA; NC, negative control.

miR-874 directly targets ETS1 in cervical cancer cells

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).

Figure 4. Identification of ETS1 as a novel target of miR-874 in cervical cancer cells. (A) Predicted wild-type (Wt) and mutant (Mut) miR-874 binding sequences in the 3′-UTR of ETS1 are shown. (B) SiHa and HeLa cells were co-transfected with miR-874 mimics or miR-NC and pGL3-ETS1-3′-UTR Wt and pGL3-ETS1-3′-UTR Mut. The luciferase activities were determined by a dual luciferase reporter assay kit at 48 h post-transfection. *P<0.05 vs. miR-NC. (C) RT-qPCR analysis was conducted to quantify the ETS1 mRNA expression in 49 pairs of cervical cancer tissues and adjacent non-cancerous tissues. *P<0.05 vs. non-cancerous tissues. (D) The correlation between the expression levels of miR-874 and ETS1 mRNA was examined using Spearman's correlation analysis. r=−0.5419, P<0.0001. (E) RT-qPCR was used to examine the effect of miR-874 overexpression on endogenous ETS1 mRNA level in SiHa and HeLa cells. (F) Western blot analysis was performed to measure ETS1 protein expression in SiHa and HeLa cells transfected with miR-874 mimics or miR-NC. *P<0.05 vs. miR-NC. ETS1, E26 transformation specific-1; miR, microRNA; Wt, wild-type; Mut, mutant; UTR, untranslated region; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

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.

Inhibition of ETS1 simulates the tumour-suppressive effects of miR-874 in cervical cancer cells

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.

Figure 5. Inhibition of ETS1 restricts the proliferation and metastasis, and promotes the apoptosis of SiHa and HeLa cells. SiHa and HeLa cells transfected with ETS1 siRNA or NC siRNA were used in the subsequent analysis. (A) Transfected cells were subjected to western blot analysis to detect ETS1 protein expression. *P<0.05 vs. NC siRNA. (B and C) Cell Counting kit-8 was used to detect the proliferation of indicated cells. *P<0.05 vs. NC siRNA. (C) Colony formation assays were utilized to examine the colony formation ability of indicated cells. *P<0.05 vs. NC siRNA. (D) Apoptosis rate was assessed using flow cytometric analysis. *P<0.05 vs. NC siRNA. (E) Migration ability was determined by Transwell migration assay. (F) Transwell invasion assay was employed to evaluate the invasion capacity of indicated cells. *P<0.05 vs. NC siRNA. ETS1, E26 transformation specific-1; siRNA, small interfering RNA; NC, negative control.

Upregulation of ETS1 reverses the biological function of miR-874 overexpression 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.

Figure 6. Reintroduction of ETS1 abolishes the tumour-suppressing roles of miR-874 in cervical cancer cells. SiHa and HeLa cells were transfected with miR-874 mimics along with pCMV-ETS1 or pCMV. At different time points, the transfected cells were used in the subsequent analysis. (A) ETS1 protein expression was measured by western blot analysis. *P<0.05 vs. miR-NC. #P<0.05 vs. miR-874 mimics+pCMV-ETS1. (B) Cell Counting kit-8 assay was performed to detect the proliferation of the abovementioned cells. (C) Colony formation assay was used to assess the colony formation ability of the aforementioned cells. (D) The apoptosis rate of the aforementioned cells was measured using flow cytometric analysis. (E) Transwell migration assay was conducted to examine the migration of the aforementioned cells. (F) The invasion capacity of the aforementioned cells was assessed using Transwell invasion assay. *P<0.05 vs. miR-NC. #P<0.05 vs. miR-874 mimics+pCMV-ETS1. ETS1, E26 transformation specific-1; miRNA, microRNA; NC, negative control.

Discussion

An increasing number of studies have reported that miRNAs are differentially expressed in cervical cancer and serve critical roles in cervical oncogenesis and progression (23–25). Therefore, identifying the aberrantly expressed miRNAs implicated in the formation and progression of cervical cancer may provide key clues for the development of effective therapeutic targets in treating patients with this malignancy. In our current study, miR-874 expression was significantly downregulated in cervical cancer tissues and cell lines. Decreased miR-874 expression was correlated with FIGO stage and lymph node metastasis. In addition, resumption of miR-874 expression suppressed the proliferation, migration and invasion, but increased the apoptosis of cervical cancer cells. Further investigation demonstrated that ETS1 is a novel target of miR-874 in cervical cancer. Furthermore, ETS1 expression was upregulated in cervical cancer tissues, and the upregulation of ETS1 was inversely correlated with miR-874 expression. Furthermore, ETS1-knockdown simulated the tumour suppressive effects of miR-874 in cervical cancer cells. Notably, reintroduction of ETS1 expression counteracted the inhibitory effects of miR-874 overexpression in cervical cancer cells. These findings suggested that miR-874 may be a valuable therapeutic target for treating patients with cervical cancer.

miR-874 is dysregulated in diverse types of human malignancy. For example, miR-874 is downregulated in colorectal cancer, and the downregulation of miR-874 is associated with TNM stage and lymph node metastasis (18). In osteosarcoma, miR-874 expression is downregulated in tumour tissues and cell lines. Low miR-874 expression is strongly correlated with TNM stage, tumour size and lymph node metastasis in patients with osteosarcoma (19). In hepatocellular carcinoma, decreased miR-874 expression in tumour tissues is significantly associated with tumour size, vascular invasion, TNM stage, tumour differentiation and inferior patient outcomes (20). In gastric cancer, miR-874 expression is reduced in clinical samples and few cell lines. The downregulation of miR-874 expression is correlated with certain characteristic features of patients with gastric cancer that indicate an unfavourable prognosis (21). These findings demonstrated that miR-874 is frequently mildly expressed in these types of human cancer and suggested that this miRNA has a strong diagnostic and prognostic value.

Dysregulation of miR-874 participates in the occurrence and development of multiple types of human cancer. For instance, miR-874 overexpression restricts colorectal cancer cell proliferation, inhibits colony formation, induces apoptosis and improves the chemosensitivity to 5-FU by directly targeting XIAP and STAT3 and inhibiting the Hippo signalling pathway (18,26,27). Dong et al demonstrated that miR-874 targets E2F3 to impede proliferation and motility, and to promote the apoptosis of osteosarcoma cells (19). Multiple studies have reported that induced expression of miR-874 suppresses the growth, metastasis and epithelial-mesenchymal transition, but promotes the apoptosis in vitro and reduces the tumourigenicity in vivo of hepatocellular carcinoma cells by inhibiting DOR, SOX12 and PIN1 expression (20,28,29). Studies by Jiang et al (21) and Zhang et al (30) revealed that miR-874 re-expression represses the angiogenesis, growth and motility in vitro, and the tumourigenicity in vivo of gastric cancer cells via the blockade of STAT3 and AQP3. These findings suggested that miR-874 may hold the potential to be developed as a therapeutic target in treating patients with these specific human malignancy types.

ETS1, a member of the ETS family of transcription factors, is validated as a direct target gene of miR-874 in cervical cancer. It directly binds to specific DNA sequences containing a GGAA/T core motif and serves crucial roles in carcinogenesis and cancer progression. ETS1 is reportedly overexpressed in multiple types of human malignant tumour, including ovarian cancer (31), colorectal cancer (32), prostate cancer (33) and gastric cancer (34). ETS1 is also upregulated in cervical cancer, and its upregulation is significantly correlated with tumour differentiation and lymph node metastasis (35). Patients with cervical cancer exhibiting high ETS1 levels exhibit a poorer prognosis than those with low ETS1 levels (35,36). The deregulated ETS1 is implicated in the oncogenesis and development of multiple types of human cancer by regulating several pathological behaviours (37–39). Taken together, these findings indicated that targeting ETS1 through miRNA-based targeted therapy has promising therapeutic applications in treating patients with cervical cancer.

In summary, to the best of our knowledge, the present study was the first to reveal that miR-874 inhibits the progression of cervical cancer by directly targeting ETS1. These findings highlighted the importance of the miR-874/ETS1 pathway in the treatment of cervical cancer in the future.

Acknowledgements

Not applicable.

Funding

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

YJ and HL designed this research. HL, YunfeiP, YuefenP, JS, QQ and LZ performed functional experiments. WH and QW analyzed the data of this study. All authors have read and approved the final draft.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Huzhou Central Hospital (Huzhou, China), and was performed in accordance with the Declaration of Helsinki and the guidelines of the Ethics Committee of Huzhou Central Hospital. Written informed consent was obtained from all patients for the use of their clinical tissues.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References