Human samples used in the present study were obtained according to the principles of the Declaration of Helsinki, and all procedures were approved by the Ethics Committee of the Renmin Hospital of Wuhan University (Wuhan, China). Written informed consent was obtained from all patients.

The study cohort consisted of 13 patients with invasive carcinoma of the cervix (ICC) at the early stage (termed stage IA-IIA) identified by pathological diagnosis (age range, 29–66 years; mean age, 46 years), who were treated at Renmin Hospital of Wuhan University (Wuhan, China) between February and July 2014. A total of 13 cervical cancer tissues and 12 normal cervical epithelial tissues were collected at the same time points. Staging was performed according to the standard of the International Federation of Gynecology and Obstetrics (FIGO) (21).

The human cervical carcinoma cell line HeLa was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HeLa cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and cultured in an incubator at 37°C with 5% CO₂. The cells were then seeded into six-well plates and grown overnight to reach ~40% confluence. Cells were transfected with miR-200b mimics (Ribobio, Guangzhou, China), miR-200b inhibitors (Ribobio) and negative control miR (Ribobio) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) in Opti-MEM (Invitrogen) according to the manufacturer's protocol. After 6 h of transfection, the medium (DMEM; Invitrogen) was removed and replaced with complete growth medium. At 48 h after transfection, cells were harvested for RNA or protein analysis.

Total RNA was extracted with TRIzol regent (Invitrogen) in accordance with the manufacturer's protocol. For analysis of miR-200b expression, cDNA was synthesized using specific reverse transcription primers (miR-200b, F 5′-GCG GCT AAT ACT GCC TGG TAA-3′; R 5′-GTG CAG GGT CCG AGGT-3′; U6, F 5′-CGC TTC GGC AGC ACA TAT ACTA-3′; R 5′-CGC TTC ACG AAT TTG CGT GTCA-3′). purchased from Ribobio and the miRNA First-strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). Real-time PCR was performed in the Step One Plus Real-Time system (Applied Biosystems, Thermo Fisher Scientific, Inc.) with ABI SYBR Green Master Mix (Applied Biosystems), and U6 was used as the endogenous reference gene. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The gene expression levels were calculated relative to the expression of U6 using the 2^−ΔΔCq method (22).

Cell migration was examined by a Transwell migration assay. Transwell insert chambers with 8-µm porous membranes (Corning Inc., Corning, NY, USA) were used. Following 24 h of transfection, the cells were dislodged from the culture vessel surface with trypsin (Gibco, Thermo Fisher Scientific, Inc.) suspended at 2×10⁵ cells/ml. For each group, 200 µl cell suspension was seeded into the upper Transwell chambers. The lower compartment contained 10% fetal calf serum (Gibco). After 24 h of incubation, cells on the upper side of the membrane were removed, followed by washing with phosphate-buffered saline (PBS) three times. The migrated cells on the bottom side of the membrane were then fixed with methanol (Goodbio Technology, Wuhan, China) for 15 min at room temperature and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in 25% methanol for 5 min. Images of migrated cells in at least six random selected fields were captured under a microscope at 100× magnification and cells were counted. Experiments were performed in technical and biological triplicates. One representative high-power field per Transwell membrane was counted (using six randomly selected high-power fields per treatment condition).

Following transfection, cells were lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The obtained protein extracts were centrifuged at 16,099 × g for 15 min. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). For western blot analysis, equal quantity (30 µg) of protein was loaded in each lane and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking, the membranes were probed with antibodies against monoclonal anti-rabbit human E-cadherin (1:1,000; cat. no. 3195), monoclonal anti-rabbit human vimentin (1:1,000; cat. no. 5741) or anti-rabbit human GAPDH (1:1,000; cat. no. 2118; Cell Signaling Technology, Inc., Danvers, MA, USA), or monoclonal anti-mouse human matrix metalloproteinase (MMP-9) antibody (1:800; cat. no. ab58803; Abcam, Cambridge, UK) overnight at 4°C. After washing in Tris-buffered saline containing Tween 20 (TBST), the membranes of E-cadherin, vimentin and GAPDH were incubated with bovine anit-rabbit horseradish peroxidase-conjugated secondary antibody (1:8,000; cat. no. sc-2370; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and MMP-9 membranes were incubated with bovine anti-mouse horseradish peroxidase-conjugated secondary antibody (1:8,000; cat. no. sc2371; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. After washing of the membranes in TBST, the proteins were visualized using an enhanced chemiluminescence detection kit (EMD Millipore) and specific antibody binding was visualized and quantified using Image J software (version 1.46; National Institutes of Health, Bethesda, MD, USA). Experiments were performed three times.

Cells grown on coverslips in a culture plate were washed three times with cold PBS and fixed with 4% paraformaldehyde for 15 min. Coverslips were then washed three times with PBS and rinsed with 0.5% Triton X-100 in PBS for 20 min. After blocking with goat serum (Goodbio Technology) for 30 min at room temperature, cells were incubated with primary antibody overnight at 4°C. Coverslips were rinsed three times with PBS containing Tween 20 (PBST) and then labeled with the secondary antibody for 1 h in the dark at room temperature. After incubation, coverslips were then washed three times with PBST and incubated with DAPI (Beyotime Institute of Biotechnology) for 5 min in the dark and washed again with PBST. Following mounting with Antifade Mounting Medium (EMD Millipore), images were acquired with a fluorescence microscope (CKX31; Olympus Corp., Tokyo, Japan).

Statistical analyses were performed with SPSS 19.0 software (International Business Machines, Armonk, NY, UA). Values are expressed as the mean ± standard deviation of at least three independent experiments. The normality test was performed using the K-S test and the homogeneity test for variance was conducted by Levene's test. Statistical analysis was performed using the independent-samples t-test (normal distribution, equal variances) and one-way analysis of variance followed by the least-significant differences test (equal variances) or Dunnett's T3 test (unequal variances) when appropriate. P<0.05 was considered to indicate a statistically significant difference between values.

To investigate the expression of miR-200b in the cervical cancer tissues and normal adjacent tissues, RT-qPCR was performed on 13 tumor specimens and 12 normal cervical epithelial samples. While miR-200b was expressed in all samples (Fig. 1A), the ICC tissues exhibited a significantly lower expression of miR-200b compared with that in normal tissues (t=7.353; P=0.002) (Fig. 1B), suggesting that miR-200b may have a tumor suppressor role in ICC.

For loss-and gain-of-function studies of miR-200b in ICC, HeLa cells were transfected with miR-200b mimics and inhibitor, and the resulting upregulation or knockdown of miR-200b gene expression were confirmed by RT-qPCR. Transfection of miR-200b mimics significantly increased the expression of miR-200b to 50 fold of that of the negative control group (P=0.007) (Fig. 2), while a significant decrease by 0.4-fold was achieved in cells transfected with inhibitor (P=0.01). These results suggested the successful overexpression and knockdown, respectively, of miR-200b.

The migratory capacity of neoplastic cells is linked with their degree of malignancy due to its association with metastasis and represents an important potential therapeutic target. Therefore, the present study examined the ability of HeLa cells transfected with miR-200b mimics or miR-200b inhibitor to migrate to the lower compartment. As shown in Fig. 3, transfection with miR-200b mimics significantly inhibited the cell migratory ability compared with that of the negative control group (P<0.001). Conversely, a significant elevation of the cell migratory ability was found in cells transfected with miR-200b inhibitor (P<0.001). These results suggested that miR-200b has an inhibitory effect on the migratory ability of ICC, explaining for the downregulation of this tumor suppressor miR in this invasive cancer type.

To test whether miR-200b affects the expression of proteins involved in the EMT, HeLa cells were transfected with miR-200b mimics or inhibitor. In cells transfected with miR-200b mimics, a substantial elevation of E-cadherin (P<0.001) and a reduction in vimentin (P=0.002) and MMP-9 (P=0.002) was observed compared to that in the negative control group (Fig. 4A–D). Conversely, inhibition of miR-200b reduced the expression of E-cadherin (P=0.002), but increased the expression of vimentin (P<0.001) and MMP-9 (P<0.001) (Fig. 4A–D). These results indicated that miR-200b was able to suppress the EMT process of HeLa cells, and to therefore potentially inhibit cervical carcinoma cell metastasis. To further verify the effects of miR-200b on the EMT process of cervical carcinoma cells, the expression of E-cadherin, which mediates cell-cell adhesion and has a pivotal role in epithelial cell behavior and tissue morphogenesis/remodelling, was determined by immunofluorescence analysis. As shown in Fig. 4E, miR-200b mimics elevated the expression of E-cadherin, while inhibition of miR-200b reduced E-cadherin. These findings provided further evidence to support the causal role of miR-200b in the EMT process of cervical carcinoma.