The present study was approved by the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China), and written informed consent was obtained from all patients. Cervical cancer and paired adjacent normal cervical tissues were collected from 53 cervical cancer patients who were treated with surgical operation between February 2011 and November 2014 at the Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Wenzhou Medical University. None of these patients had received radiotherapy or chemotherapy prior to surgery. All fresh tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C until use.

The human cervical cancer cell lines (HeLa, C33A, Caski and SiHa), an immortalized HPV-negative skin keratinocyte line (HaCaT) and the HEK293T cell line were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA) in a humidified atmosphere containing 5% CO₂ and 100% humidity at 37̊C.

Total RNA was extracted form tissues and cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription was performed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Detection and quantitation of miR-329-3p and MAPK1 mRNA were performed using SYBR Premix Ex Taq™ kits (Takara, Tokyo, Japan) on Applied Biosystems^® 7900HT Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). U6 snRNA and GAPDH were used as reference genes for miR-329-3p and MAPK1 mRNA expression, respectively. The relative expression levels of miR-329-3p and MAPK1 mRNA were analyzed using the 2^−ΔΔCt method.

The miR-329-3p and corresponding negative control mimics (miR-NC) were obtained from GenePharma (Shanghai, China). Small interfering RNA targeting MAPK1 (si-MAPK1) and its negative control (si-NC) were purchased from Ambion (Shanghai, China). The overexpression plasmid of MAPK1 (pCDNA3.1-MAPK1) and blank plasmid (pCDNA3.1) were synthesized at the Chinese Academy of Sciences (Changchun, China). For transfection, the cells were seeded in a 6-well plate until reaching 50–60% confluency. The following day, the cells were transfected with the mimics, siRNA or plasmid using Lipofectamine™ 2000 reagent (Thermo Fisher Scientific) following the manufacturer's instructions.

Cell proliferation was evaluated using the CCK-8 assay (Dojindo, Kumamoto, Japan). Briefly, the transfected cells were harvested, suspended and seeded in 96-well plates at a density of 3,000 cells/well. Cells were incubated in a humidified atmosphere containing 5% CO₂ and 100% humidity at 37̊C for 4 consecutive days after seeding. At each time point, 10 µl CCK-8 solution was added in each well for another 4-h incubation at 37̊C. Finally, the absorbance was determined at a wavelength of 450 nm using a microplate reader (Infinite^® M1000 PRO; Tecan, Männedorf, Switzerland).

Migration assays were performed using Transwell chambers (8-µm; BD Biosciences, Franklin Lakes, NJ, USA). After transfection for 48 h, the cells were trypsinized, washed with PBS and re-suspended in FBS-free DMEM. Then, 5×10⁴ cells were seeded in the upper part of each Transwell chamber, while the lower part of each Transwell chamber was filled with 600 µl DMEM containing 20% FBS. After incubation for 48 h in a humidified atmosphere containing 5% CO₂ and 100% humidity at 37̊C, the cells migrating to the bottom of the Transwell membrane were fixed with 100% methanol, stained with 0.5% crystal violet solution, dried in air and photographed under a microscope (Olympus, Tokyo, Japan). Invasion assays were carried out in a similar manner but by allowing the cells to migrate through Matrigel (BD Biosciences, San Jose, CA, USA)-coated Transwell chambers.

TargetScan Human 7.0 (http://www.targetscan.org/) and miRanda (http://www. microrna.org/microrna/) were used to identify the potential target genes of miR-329-3p.

To explore whether MAPK1 was a direct target gene of miR-329-3p, luciferace reporter assay was performed. For the luciferase reporter assay, luciferase reporter plasmids (pmirGLO-MAPK1–3′UTR Wt and pmirGLO-MAPK1–3′UTR Mut) were synthesized and purified by GenePharma. HEK293T cells were seeded in triplicate in 24-well plates. After incubation overnight, the cells were transfected with luciferase reporter plasmids, along with miR-329-3p mimics or miR-NC using Lipofectamine 2000. Transfected cells were collected 48 h post-transfection, and luciferase activities were detected using Dual-Luciferase Reporter Assays (Promega, Manheim, Germany) following manufacturer's procedures. Renilla luciferase activities were measured as a control.

For western blotting, total protein was extracted from tissues and cells using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate and 1% SDS) supplemented with proteinase and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of proteins were separated using 10% SDS polyacrylamide gels. The separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) containing 5% skimmed milk at room temperature for 1 h. Then, the PVDF membranes were incubated with primary antibodies at 4̊C overnight. Next, the membranes were washed with TBST 3 times and probed with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Finally, the protein bands were visualized using ECL chemiluminescence reagents (Amersham Biosciences Corp., Piscataway, NJ, USA). Primary antibodies used in the present study included, mouse anti-human monoclonal MAPK1 antibody (1:1,000 dilution; sc-81459) and mouse anti-human monoclonal GAPDH antibody (1:1,000 dilution; sc-137179) (both from Santa Cruz Biotechnology). GAPDH was used as an internal control.

Data are expressed as mean ± SD, and the Student's t-test was used to compare differences between two groups. P-value of <0.05 was considered to indicate a statistically significant result. All analyses were carried out using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA).

To investigate whether or not miR-329-3p is abnormally expressed in cervical cancer, we analyzed its expression in cervical cancer and paired adjacent normal cervical tissues using RT-qPCR. The results showed that miR-329-3p was significantly downregulated in the cervical cancer tissues compared with that in the paired adjacent normal cervical tissues (Fig. 1A; P<0.05).

We next analyzed the correlation between miR-329-3p expression levels and clinicopathological characteristics of the cervical cancer patients. The correlations between miR-329-3p expression levels and the clinicopathological characteristics of the cervical cancer patients are shown in Table I. The results showed that miR-329-3p was inversely correlated with histological grade (P=0.037), International Federation of Gynecology and Obstetrics (FIGO) stage (P=0.024) and lymph node metastasis (P=0.007). However, there were no significant association between miR-329-3p expression and age (P=0.269), tumor size (P=0.200), family history of cancer (P=0.504) and distant metastasis (P=0.707).

Further experiments were carried out using an immortalized HPV-negative skin keratinocyte line (HaCaT) and 4 cervical cancer cell lines to confirm that expression levels of miR-329-3p were reduced in cervical cancer cell lines, including HeLa, C33A, Caski and SiHa cells in comparison with HaCaT (Fig. 1B; P<0.05). Taken together, these results indicated that miR-329-3p was lowly expressed in the cervical cancer tissues and cell lines.

To further explore the roles of miR-329-3p in cervical cancer, miR-329-3p mimics were used to increase its expression in HeLa and SiHa cells (Fig. 2A; P<0.05). CCK-8, and migration and invasion assays were performed to test the effects of miR-329-3p overexpression on cell proliferation, migration and invasion of cervical cancer, respectively. As shown in Fig. 2B, upregulation of miR-329-3p obviously inhibited the proliferation of HeLa and SiHa cells. The results of the migration and invasion assays showed that the migration and invasion capacities of the HeLa and SiHa cells were reduced when cells were transfected with miR-329-3p mimics (Fig. 2C; P<0.05). These results indicated that miR-329-3p re-expression inhibited cell proliferation, migration and invasion of cervical cancer.

To explore the mechanism underlying the tumor-suppressive roles of miR-329-3p in cervical cancer, we next aimed to explore the potential targets of miR-329-3p. Bioinformatic analysis was performed with publicly available algorithms to predict the candidate targets of miR-329-3p. As shown in Fig. 3A, 3′UTR of MAPK1 contains a target sequence for miR-329-3p. Following, a luciferase reporter assay was carried out to further confirm whether MAPK1 is a direct target of miR-329-3p. HEK293T cells were co-transfected with pmirGLO-MAPK1–3′UTR Wt or pmirGLO-MAPK1–3′UTR Mut, and miR-329-3p mimics or miR-NC. Results showed that miR-329-3p overexpression significantly decreased luciferase activities in the HEK293T cells transfected with pmirGLO-MAPK1–3′UTR Wt, but no significant change in cells with pmirGLO-MAPK1–3′UTR Mut were noted (Fig. 3B; P<0.05). Moreover, RT-qPCR and western blotting were adopted to determine the regulatory roles of miR-329-3p on MAPK1 expression. As shown in Fig. 3C and D, restoration of the expression of miR-329-3p obviously downregulated MAPK1 expression in the HeLa and SiHa cells at the mRNA (P<0.05) and protein (P<0.05) levels. Taken together, these results demonstrated that MAPK1 is directly targeted by miR-329-3p.

The above results indicated that MAPK1 is a direct target of miR-329-3p; therefore, we next analyzed the expression of MAPK1 in cervical cancer and paired adjacent normal cervical tissues. Results of RT-qPCR revealed that MAPK1 mRNA was significantly upregulated in cervical cancer tissues compared with that noted in the paired adjacent normal cervical tissues (Fig. 4A; P<0.05). Moreover, Spearman's correlation analysis showed a negative correlation between miR-329-3p and MAPK1 mRNA expression levels in the cervical cancer tissues (Fig. 4B; r=−0.5598; P<0.001). Moreover, MAPK1 protein expression in the cervical cancer and paired adjacent normal cervical tissues was determined using western blotting. As shown in Fig. 4C, MAPK1 protein was highly expressed in the cervical cancer tissues when compared with that in the paired adjacent normal cervical tissues (P<0.05).

To confirm that the tumor-suppressive roles of miR-329-3p are mediated by downregulation of MAPK1, we investigated the biological roles of MAPK1 in cervical cancer. si-MAPK1 was employed to knock down MAPK1 expression in the HeLa and SiHa cells (Fig. 5A; P<0.05). Results of the CCK-8 assay showed that downregulation of MAPK1 obviously suppressed the proliferation of the HeLa and SiHa cells which was similar to the effect of miR-329-3p overexpression on cell proliferation (Fig. 5B; P<0.05). In addition, the effects of MAPK1 underexpression on migration and invasion of HeLa and SiHa cells were similar to those induced by miR-329-3p overexpression (Fig. 5C; P<0.05). These results indicated that restoration of the expression of miR-329-3p suppressed cell proliferation, migration and invasion of cervical cancer through downregulation of MAPK1.

Rescue experiments were performed to further confirm that MAPK1 is a direct and functional downstream target of miR-329-3p. pcDNA3.1-MAPK1 was used to increase MAPK1 expression in the HeLa and SiHa cells (Fig. 6A; P<0.05). Notably, restoration of the expression of MAPK1 significantly reversed the inhibition of HeLa and SiHa cell proliferation (Fig. 6B; P<0.05), migration and invasion (Fig. 6C; P<0.05) induced by miR-329-3p overexpression. These results indicated that miR-329-3p targets MAPK1 directly, resulting in inhibition of cell proliferation, migration and invasion of cervical cancer.