The tissue-based specimen collection and study were approved by the Research Ethics Committee of Xi’an Jiaotong University. All the patients provided written consent and indicated willingness to donate their blood and tissue samples. A total of 89 patients were enrolled in the present study. Clinical and pathological classification and staging were performed according to the International Federation of Gynecology and Obstetrics criteria (21). The clinicopathological information of the patients is shown in Table I. The follow-up information for all participants was updated every 3 months by telephone. Information regarding the death of patients was ascertained from their family. In all 89 snap-frozen cervical cancer samples, the HC2 assay was used to detect the presence of high-risk HPV DNA, including DNA from HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 (22). High-risk HPV (HR-HPV) was detected in 79 cases, which gave an overall infection rate of 88.8%.

Primary normal cervical epithelial cells (NCEC) obtained from healthy female cervical tissue were cultured in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA, USA) supplemented with epithelial growth factor, bovine pituitary extract and antibiotics (1% streptomycin and 1% penicillin). The HeLa, C33A, Caski and SiHa cervical cancer cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen). The cells were supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% penicillin/streptomycin (Invitrogen).

The expression of miR-494 in cervical cancer and corresponding adjacent tissues was detected by the RT-qPCR assay. Briefly, total RNA was extracted from tissues using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. miRNA expression levels were quantified using a TaqMan miRNA real-time RT-PCR kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Data were analyzed using 7500 software v. 2.0.1 (Applied Biosystems), with the automatic Ct setting for adapting the baseline and threshold for Ct determination. The universal small nuclear RNA U6 (RNU6B) was used as an endogenous control for miRNAs. Each sample was examined in triplicate and the amount of PCR products produced was non-neoplasticized to RNU6B.

miR-494 inhibitors were chemically synthesized by Shanghai GenePharma (GenePharma, Shanghai, China). When the cells reached 80% confluence, miR-494 inhibitor was transfected into cervical cancer cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were also transfected with scramble oligonucleotide as a negative control (NC). The expression level of mir-494 in the transfected osteosarcoma cells were identified by RT-qPCR.

Cervical cancer cells were seeded in 96-well plates at 60% confluence. After 24 h, the cells were transfected with 120 ng of miR-494 expression vector or NC. The cells were transfected with 30 ng of wild-type (WT) or mutant (MT) 3′-UTR of PTEN mRNA. The cells were collected 48 h after transfection, and luciferase activity was measured using a dual luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Cells were plated in 96-well plates (0.5×10⁴ cells/well) and transfected with NC, and miR-494 inhibitors. After 48 h, 10 μl of MTT reagent (5 mg/ml) was added to each well and the cells were incubated at 37°C for another 4 h. The medium was removed, the cells were solubilized in 150 μl of dimethyl sulfoxide, and colorimetric analysis was performed (wavelength, 490 nm). One plate was analyzed immediately after the cells adhered (~4 h after plating), and the remaining plates were assayed every day for the following 4 consecutive days.

Briefly, 10 cm dishes were seeded with 500 viable cells in complete medium and allowed to grow for 24 h. The cells were then incubated in the presence of miR-494 inhibitors or NC for up to 48 h. The medium was removed, and the cells were washed in phosphate-buffered saline (PBS) and incubated for an additional 10 days in complete medium. Each treatment was carried out in triplicate. The colonies obtained were washed with PBS and fixed in 4% formalin for 10 min at room temperature and then washed with PBS followed by staining with 0.2% crystal violet.

Cells seeded in a 6-well plate were covered with a layer of 0.6% agar in DMEM medium supplemented with 10% FBS. After transfection for 48 h, the cells were trypsinized, gently mixed with 0.3% agar medium mixture containing selective antibiotics and reseeded in triplicate in a 6-well plate. After 4 weeks, the resistant colonies were stained with 0.2% crystal violet and counted under the microscope.

The cervical cancer cells were transfected with NC and miR-101 inhibitors. Forty-eight hours after post-transfection, the cells were trypsinized and analyzed for cell cycle distribution. For cell cycle distribution, the cells of each group were stained with propidium iodide (PI) and analyzed by flow cytometry using FACSCalibur (BD Biosciences, San Diego, CA, USA). For each group, 10,000 events were obtained. The percentage of cells in G1, S and G2 phases of the cell cycle was calculated.

Data are presented as mean ±SD. Statistical analysis was performed using IBM SPSS statistical software (version 21.0) (International Business Machines Corporation, Armonk, NY, USA). The differences in characteristics between the two groups were examined by the χ² or Fisher’s exact tests. P-values were determined from two-sided tests, and statistical significance was based on a P-value of 0.05.

To examine the levels of miR-494 expression in cervical cancer, we conducted RT-qPCR to measure miR-494 expression in four cervical cancer cell lines and NCEC. The result showed that miR-494 was markedly increased in the Caski, HeLa, C33A and SiHa cervical cell lines, particularly in HeLa and C33A, compared with NCEC (Fig. 1A). Consistent with the results found in cervical cell lines, miR-494 expression was significantly higher in 89 cervical cancer tissue specimens compared with their adjacent normal tissues (Fig. 1B). By contrast, the expression of PTEN was significantly down-regulated in cervical cancer tissues compared with their normal tissue counterparts (Fig. 1C), which was consistent with previous literature (23). More importantly, statistically significant inverse correlations were revealed by Spearman’s correlation analysis between mRNA levels of miR-494 and PTEN in cervical cancer specimens (r=−0.3285; P=0.0017). Taken together, the results suggested that miR-494 played an oncogenic role and PTEN a tumor-suppressor role in cervical cancer. Furthermore, miR-494 inversely correlated with PTEN in cervical cancer, which indicated that PTEN was a potential target of miR-494 in cervical cancer.

To explore the relationship between miR-494 and cervical cancer, we investigated the correlation of miR-494 expression with metastasis and recurrence of cervical cancer. Compared with non-metastatic cervical cancer specimens, the miR-494 levels were significantly upregulated in metastatic cervical tissues (Fig. 2A). Moreover, miR-494 levels were significantly higher in the specimens obtained from the patients who suffered cervical cancer recurrence (Fig. 1B). Collectively, these data indicated that significantly upregulation of miR-494 expression was correlated with relapse and metastasis in cervical cancer patents.

In order to determine the clinical significance of miR-494 in cervical cancer, the 89 patients were divided into two groups based on miR-494 expression levels (low vs. high) with the median expression levels as a cut-off point. The Kaplan-Meier analysis revealed that high miR-494 expression was significantly correlated with reduced overall and progression-free survival in 89 cervical cancer patients (Fig. 2C and D; log-rank test, P=0.0104 and P=0.0123, respectively). The patients with a high miR-494 expression tended to have a shorter overall and progression-free survival time when compared to patients with a low miR-494 expression. In addition, upregulation of miR-494 was significantly correlated with FIGO stage, lymph-node metastasis and deep stromal invasion while no significant correlation was observed in other clinicopathological variables (Table I). The, univariate analysis demonstrated that the overall and progression-free survival of cervical cancer patients was associated with FIGO stage, lymph-node status, and HR-HPV and miR-494 expression (Tables II and III). To determine whether the prognostic value of miR-494 was independent of other clinicopathological parameters for poor overall and progression-free survival in cervical cancer patients, a multivariate analysis was performed using a Cox proportional hazard model. The multivariate analysis including miR-494 expression, age, FIGO stage, HR-HPV, differentiation status, tumor size and lymph-node metastasis demonstrated that a high miR-494 expression was an independent prognostic biomarker for poor overall and progression-free survival in cervical cancer patients (Tables II and III; HR=3.279, CI=1.177–5.192, P=0.013 and HR=4.614, CI=2.895–10.321, P<0.001 respectively). Statistically significant results were also obtained for FIGO stage and lymph-node metastasis, where the other parameters were not independent prognostic biomarkers for overall and progression-free survival in cervical cancer patients. Taken together, these results suggest the upregulation of miR-494 was significantly correlated with a worse prognosis and was involved in the progression of cervical cancer.

As the relative expression of miR-494 was relatively higher in HeLa and C33A than SiHa and Caski, we chose HeLa and C33A to investigate the physiological function of miR-494 in cervical cancer cells. To analyze the effect of miR-494 on the proliferation of cervical cancer cells, we transfected miR-494 inhibitors into HeLa and C33A cell lines. As shown in Fig. 3A, transfection of miR-494 inhibitors decreased the miR-494 expression in HeLa and C33A (Fig. 3A). After confirming the efficiency of miR-494 inhibitors, we determined the effects of miR-494 on cell viability using an MTT assay. Cervical cancer cells transfected with miR-494 inhibitors showed a significant decrease in cell viability as compared with the normal control (Fig. 3B). We determined the effect of miR-494 on cell proliferation using colony formation and soft agar colony formation assays. As shown in Fig. 4C and D, inhibition of miR-494 significantly decreased the growth rate of the two cervical cell lines as compared with the normal control (Fig. 4C and D). Taken together, these results indicated that the downregulation of miR-494 suppressed the proliferation of cervical cancer cells.

As miR-494 significantly affects cell proliferation in HeLa and C33A cells, we hypothesized that miR-494 functions by affecting the cell cycle of cervical cancer cells. Thus, we investigated the effect of miR-494 on the cell cycle by flow cytometry. The results revealed that overexpression of miR-494 inhibitors markedly increased the number of cells in G1 peak and decreased those in the S peak (Fig. 3E and F). Taken together, these results indicated the inhibition of miR-494 suppressed the proliferation of cervical cancer cells by inducing cell cycle arrest.

As overexpression of miR-494 inhibitors appears tobe closely linked to the proliferation of cervical cancer cells, we further investigated whether the CDK inhibitor p21^Cip1 or the CDK regulator cyclin D1 could be regulated by miR-494. RT-qPCR and western blot analysis revealed that p21^Cip1 was upregulated, whereas cyclin D1 was downregulated in cervical cells transfected with miR-494 inhibitors compared with cells transfected with the normal control (Fig. 4A and B). Taken together, these results supported our hypothesis that miR-494 has a critical role in the growth of cervical cancer cells.

It has been proven that PTEN was the direct target of miR-494 in multiple solid tumors (24), and loss of protein expression of PTEN was involved in the pathogenesis, proliferation and metastasis of cervical cancer (25,26). Considering the tissue-specific and developmental stage-specific manner of miRNA, we investigated the relationship between PTEN and miR-494 in cervical cancer. In order to confirm PTEN is the target gene for miR-494 in cervical cancer cells, RT-qPCR and western blotting was used to detect the expression of PTEN in HeLa and C33A. As expected, the expression of PTEN at the mRNA and protein level was significantly upregulated in cervical cancer cells transfected with miR-494 inhibitors (Fig. 4C and D). Our previous results demonstrated the mRNA of PTEN was inversely correlated with miR-494 expression (Fig. 1D). Taken together, these results suggested that PTEN was the potential target gene of miR-494 in cervical cancer cell lines and tissues.

Then we performed the luciferase reporter assay to further verify whether miR-494 directly targeted the 3′-UTR of PTEN in cervical cancer cells. The target sequence of wild-type PTEN 3′-UTR (WT 3′-UTR) or the mutant PTEN 3′-UTR (MT 3′-UTR) was cloned into a luciferase reporter vector (Fig. 4E). As shown in Fig. 4F, transfection of miR-494 consistently suppressed the luciferase activity of PTEN WT3′UTR luciferase reporter plasmids in HeLa and C33A cells, whereas point mutations in the miR-494-binding seed region of the PTEN abrogated the repressive effect of miR-494. Taken together, the data suggested that PTEN was a genuine target of miR-494.