A total of 60 human-derived CC specimens and non-cancerous tissues were sampled between 2015 and 2017 at The Second Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine (Shaanxi, China). Of the patients involved, 33 were male and 27 were female, aged between 42 and 63 years of age. The experiments were approved by the Ethics Committee of The Second Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine. All tissues were collected prior to the initiation of radiotherapy or chemotherapy and were frozen immediately and stored at -80°C until required. Cytological and/or pathological evidence of a CC diagnosis were available from each subject. Patients provided written informed consent.

The Ect1/E6E7 human normal cervical cell line, CC cell lines of human origin (HeLa, SiHa, Caski, Me180, Ms751 and C33a) and 293T cells were procured from the American Type Culture Collection (Rockville, MD, USA). The cells were grown in RPMI 1640 medium or Dulbecco's modified Eagle's medium (DMEM, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified chamber with 5% CO₂ at 37°C.

The miR-217-mimic, miR-217-negative control (NC) and MAPK1 expression vector were purchased from GenePharma (Shanghai, China). Lipofectamine^® 3000 transfection reagent (Thermo Fisher Scientific, Inc.) was used to transfect the cells with the aforementioned miRNAs according to the manufacturer's protocol, with a transfection incubation time of 6 h. PD98059 was obtained from Tocris Bioscience (Ellisville, MO, USA; CAS no. 167869-21-8, HPLC ≥98%).

A Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Beijing, China) was performed to assess cell viability according to the manufacturer's protocol. Briefly, the transfected and untransfected cells were transferred to 96-well plates (3,000 cells/well). Following incubation for 2 h in 10 μl CCK-8 at 37°C, the absorbance in every well was read at 450 nm using a microplate reader (Tecan Infinite M200 Microplate Reader; LabX, Tecan Group, Ltd., Männedorf, Switzerland).

The potential target genes of miR-217 were predicted using TargetScan 7.2 online software (www.targetscan.org) according to the manufacturer's instructions. 'miR-217' was inserted and 'human' was selected. The putative target genes of miR-217 were scanned.

Wild-type MAPK1 (MAPK1-WT) and mutated MAPK1 (MAPK1-MUT) were cloned into pMIR-REPORT luciferase vectors (Ambion; Thermo Fisher Scientific, Inc.). 293T cells (5×10⁵ cells/well) were seeded into 6-well plates and then transfected with both vectors using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) for 24 h, according to the manufacturer's manual. The Dual-Luciferase-Reporter 1000 Assay system (Promega Corporation, Madison, WI, USA) was used to assess luciferase activity. Renilla activity was used for normalization.

The total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The concentration and purity of RNA were determined using the NanoDrop2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). The total RNA (1 μg) was reverse transcribed using a reverse transcription cDNA kit (Thermo Fisher Scientific, Inc.) and TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific, Inc.) for the synthesis of cDNA (42°C for 60 min, 70°C for 5 min, preserved at 4°C). SYBR-Green PCR Master mix (Roche Diagnostics, Basel, Switzerland) and the TaqMan miRNA PCR kit (Applied Biosystems, Thermo Fisher Scientific, Inc.) were used to perform qPCR assays for MAPK1 and miRNA-217 using the Opticon RT-PCR detection system (ABI 7500, Thermo Fisher Scientific, Inc.), The PCR cycle was set as follows: Pretreatment at 95°C for 10 min, followed by 40 cycles at 94°C for 15 sec, 60°C for 1 min, 60°C for 1 min and preserved at 4°C. The comparative quantification cycle (2^−ΔΔCq) method (21) was used to analyze the expression of mRNA. The expression of GAPDH was used for normalization. The sequences of the primers used were as follows: MAPK1, forward 5′-GTCGCCATCAAGAAAATCAGC-3′, and reverse 5′-GGAAGGTTTGAGGTCACGGT-3′; GAPDH, forward 5′-AGAAGGCTGGGGCTCATTTG-3′, and reverse 5'-AGGGGCCATCCACAG TCTTC-3′; miR-217, forward 5′-TACTCAACTCACTACTGCATCAGG A-3′, and reverse 5′-TATGGTTGTTCTGCTCTCTGTGTC-3′; and U6, forward 5′-CTCGCTTCGGCAGCACA-3′, and reverse 5′-TGGTGTCGTGGAGTCG-3′.

Total proteins were collected using RIPA (Cell Signaling Technology, Inc., Danvers, MA, USA). A BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to measure the concentrations of proteins and adjusted to a concentration of 6 μg/μl using 1X loading and DEPC water. The samples (5 μl) were separated on 10% SDS-PAGE gels and then transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking in 5% non-fat milk in PBST [0.1% Tween-20 in phosphate-buffered saline (PBS)] for 1 h, the membrane was probed with the primary antibody overnight at 4°C and washed three times in PBST. The membrane was then incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG, 1:2,000; cat. no. sc-516102/sc-2357; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) at room temperature for 2 h. The membrane was then washed with PBST three times. A developer (EZ-ECL kit; Biological Industries) was used for development, and the gray values of the strips were analyzed and counted using ImageJ software (version 5.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The antibodies used were as follows: Anti-GAPDH (mouse; 1:1,000; cat. no. LS-B1625; LifeSpan BioSciences, Inc.), anti-Bax (rabbit; 1:1,000; cat. no. ab32503; Abcam), anti-Bcl-2 (rabbit; 1:1,000; cat. no. ab32124; Abcam), anti-cleaved caspase-3 (rabbit; 1:1,000; cat. no. #9661; Cell Signaling Technology, Inc.), anti-p-ERK1/2 (rabbit; 1:1,000; cat. no. #4370; Cell Signaling Technology, Inc.), anti-ERK1/2 (mouse; 1:1,000; cat. no. #4696; Cell Signaling Technology, Inc.) and anti-MAPK1 (rabbit; 1:1,000; cat. no. #9108; Cell Signaling Technology, Inc.).

Flow cytometry was used for the analysis of apoptosis. The HeLa cells were transfected with an expression vector or mimics and incubated for 24 h. The supernatant was collected in a 15-ml centrifuge tube, and the culture flask was gently washed once by adding 2 ml PBS. Trypsin (1 ml) without ehylenediaminetetraacetic acid was used to digest the cells by shaking gently, and the pancreatic enzyme was aspirated when the wall was wet. The mixture was stood at room temperature for 1 min, and DMEM (Corning Incorporated) containing 10% FBS was added to terminate digestion. The cells were centrifuged at 1,000 × g for 3 min at 4°C, and the supernatant was removed. The cells were then washed twice with pre-cooled PBS and resuspended in 1X Annexin V binding buffer. According to the Annexin-V-FITC cell apoptosis detection kit (cat. no. K201-100, BioVision, Inc., Milpitas, CA, USA), at room temperature, the cells were collected and stained with Annexin V-FITC and propidium iodide (PI) for 15 min and counted by flow cytometry (version 10.0, FlowJo, FACS Calibur™, BD Biosciences, Franklin Lakes, NJ, USA). The flow cytometry scatter diagrams showed that living cells shown in the lower left quadrant are mechanically damaged and necrotic cells in the left upper quadrant are necrotic, whereas advanced apoptotic cells are shown in the upper right quadrant and early apoptotic cells are shown in the lower right quadrant.

Cell cycle analysis was performed by flow cytometry. To be more specific, 5×10⁵ cells with 70% cold ethanol were cultured -20°C overnight. The following day, the fixed cells were centrifuged at 1,200 × g for 1 min at 4°C, and washed twice with PBS. The cells were treated with 200 μl RNase A (1 mg/ml) for 10 min at 37°C in suspension, following which 300 μl PI (100 μl/ml, BioVision, Inc.) was added to stain the DNA in cells in the dark. Following incubation at room temperature for 20 min, the cellular DNA content of the cells was analyzed in a FACScan flow cytometer (BD Biosciences) using ModFit LT software V2.0 (BD Biosciences).

Following transfection of the HeLa cells for 24 h, a gap was created using a 200-μl sterile tip across the middle of the well. The cells were washed twice with DMEM for smoothing the edges of the scratch and floating cells were removed. Following incubation in an incubator (37°C, 5% CO₂) for 0 and 24 h, the migration of the cells was observed under a light microscope (Keyence Corporation, Osaka, Japan), the distance of cell migration was visualized and images were captured using Image-Pro Plus Analysis software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

An 8-μm Transwell chamber (cat. no. 3413, Corning Incorporated,) was placed on a 24-well plate with a layer of Matrigel (50 μl; BD Biosciences) coated on the Transwell chamber. The HeLa cells were cultured in serum-free medium for 12 h to eliminate the effects of the serum and then resuspended in DMEM containing bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) with free FBS. The suspended cells (100 μl) were added to the Transwell chamber, and 400 μl of DMEM containing 20% FBS was added to the basolateral chamber. The cells were cultured for 24 h at 37°C in an incubator with 5% CO₂. The Transwell chamber was then removed, the culture solution in the Transwell was discarded and washed twice with calcium-free PBS, and the chamber was fixed in methanol solution for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. Subsequently, the chamber was washed with PBS and the upper chamber liquid was aspirated. The unmigrated cells in the upper layer were gently wiped off using a cotton swab. The microporous membrane was carefully removed using a small pair of tweezers, dried with the bottom side facing upwards and then transferred onto a glass slide and sealed using a neutral gum. Images were observed and collected using an inverted optical microscope (Keyence Corporation).

All experimental data are presented as the mean ± SEM. P<0.05 was considered to indicate a statistically significant difference. Statistical significance was determined by one-way analysis of variance between groups, followed by a Bonferroni test. All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc.).

The mRNA levels of miR-217 in cancer tissues from patients with CC, CC cell lines and controls were measured, and the mRNA levels of MAPK1 were determined in CC tissues. The results showed that the mRNA level of miR-217 was decreased (Fig. 1A) whereas that of MAPK1 (Fig. 1B) was increased in the cancer tissue group, compared with levels in the normal group. It was also found that there was a negative correlation between the levels of miR-217 and MAPK1 (Fig. 1C). It was also found that the expression of miR-217 was lower in the CC cell lines than that in the control cells (Fig. 1D). The expression of miR-217 exhibited a greater degree of reduction in the HeLa cells, thus, the HeLa cells were used in later experiments.

To further investigate the role of miR-217 in CC, the miR-217 mimic was transfected into HeLa cells. As shown in Fig. 2A, the cells were successfully transfected with the miR-217 mimic. It was found that the miR-217 mimic suppressed cell viability (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D), compared with levels in the blank and mimic control groups, respectively.

The results revealed that apoptosis (Fig. 3A) was induced and cell cycle (Fig. 3B) was suppressed by the miR-217 mimic, compared with observations in the blank and mimic control groups, respectively. The protein level of anti-apoptotic Blc-2 was lower, whereas the expression levels of pro-apoptotic proteins (Bax and cleaved caspase-3) were higher in the miR-217 mimic group compared with those in the blank and mimic control groups (Fig. 3C and D).

To determine the binding capacity of miR-217 to MAPK1, the TargetScan 7.2 website was used and MAPK1 was identified as a potential target of miR-217 (Fig. 4A). The dual-luciferase reporter gene assay data demonstrated that the luciferase activity was lower in the MAPK1-WT mimic group than that in the MAPK1-WT blank group (Fig. 4B), with no significant changes shown in the MAPK1-MUT group.

The role of MAPK1 in CC was investigated in vitro. It was found that MAPK1 was successfully transfected into cells through observation at the protein level (Fig. 5A and B). The mRNA expression of MAPK1 was enhanced and confirmed by RT-qPCR analysis (Fig. 5C). The miR-217 mimic was found to inhibit cell viability (Fig. 5D), migration (Fig. 5E) and invasion (Fig. 5F), which were reversed by MAPK1. The miR-217 mimic-induced cell apoptosis was ameliorated by MAPK1 (Fig. 5G). The protein level of p-ERK1/2 was lower in the mimic group than that in the blank group, which was also reversed by MAPK1 (Fig. 5H and I).

p-ERK1/2 is an important component of the MAPK signaling pathway. In the present study, western blotting was performed to detect the levels of p-ERK1/2 and MAPK1, and it was found that the levels of MAPK1 and p-ERK1/2 were higher in the mimic MAPK1 group compared with those in the Blank group. However, PD98059 reversed the effects of MAPK1 on the levels of MAPK1 and p-ERK1/2 (Fig. 6A and B).