This study was approved by the Ethics Committee of Beijing Chao-Yang Hospital, and was conducted following the principles laid down in the Helsinki Declaration. Fifty-eight pairs of cervical cancer tissues and matched adjacent normal tissues were collected from patients with cervical cancer who were operated at Department of Obstetrics and Gynecology, Beijing Chao-Yang Hospital. All tissues were frozen in liquid nitrogen and stored at −80°C until use.

Human cervical cancer cell lines (HeLa, CaSki, C33A, SiHa) and an immortalized HPV-negative skin keratinocyte line (HaCaT) were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China), and grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) or RPMI-1640 medium (Gibco) containing 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). These cells were maintained at 37°C in an atmosphere containing 5% CO₂ and 100% humidity.

The miR-362 mimic, negative control (NC), SIX1 siRNA and NC siRNA were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). SIX1 overexpressed plasmid (pCDNA3.1-SIX1) and blank plasmid (pCDNA3.1) were synthesized by Chinese Academy of Sciences (Changchun, China). Transfection and co-transfection of these oligonucleotides were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Total RNA was isolated from tissues or cells by using TRIzol reagent (Invitrogen). RNA concentration was detected with NanoDrop ND-1000 spectrophotometer (Fisher Scientific, Madrid, Spain). cDNA was synthesized from total RNA using M-MLV (Promega, Madison, WI, USA). The relative expression of miR-362 and SIX1 mRNA was determined using SYBR Premix Ex Taq™ kits (Takara, Tokyo, Japan), with U6 and GADPH as endogenous control, respectively. The primers used in this study are shown in Table I. Relative expression was calculated using the 2^−∆∆Ct method.

Cell proliferation was assessed by using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) assay. Transfected cells were collected and seeded in 96-well plates at a density of 2,000 cells per well. Cells were then incubated at 37°C in an atmosphere containing 5% CO₂ and 100% humidity for 24, 48, 72, and 96 h. At indicated time-points, MTT assay was performed. In brief, 20 µl MTT solution (5 mg/ml) was added to each well and subsequently incubated at 37°C for additional 4 h. The culture medium containing MTT solution was discarded carefully, and 200 µl dimethyl sulfoxide (DMSO; Sigma) was added to solubilize the formazan precipitates. The optical density (OD) at 490 nm was measured using an automatic multi-well spectrophotometer (Bio-Rad, Richmond, CA, USA).

Transwell chambers (8-µm; Millipore, Billerica, MA, USA) and Matrigel (BD Biosciences, San Jose, CA, USA) coated Transwell chambers were adopted to perform cell migration and invasion assay, respectively. Transfected cells (5×10⁴) in FBS-free culture medium were seeded into the upper chambers. The lower chamber was filled with culture medium containing 20% FBS. Cells were incubated at 37°C in an atmosphere containing 5% CO₂ and 100% humidity for 48 h. Cells migrating to the lower surface of the Transwell membrane were fixed with 100% methanol, stained with 0.5% crystal violet solution and washed with phosphate-buffered saline (PBS; Gibco). A light microscope (Olympus IX53; Olympus, Tokyo, Japan) was used to capture five fields per membrane. Each experiment was repeated at least three times.

The potential target genes of miR-362 were predicted using the TargetScan (http://www.targetscan.org) and miRanda (http://www.microrna.org/microrna/).

PGL3-SIX1-3′UTR Wt and PGL3-SIX1-3′UTR Mut were synthesized and confirmed by GenePharma. HEK293T cells were seeded into 24-well plates, after which they were transfected with miR-362 mimics or NC, along with PGL3-SIX1-3′UTR Wt or PGL3-SIX1-3′UTR Mut using Lipofectamine 2000. Forty-eight hours after transfection, the cell lysates were subjected to luciferase activity measurement by using Dual-Luciferase Reporter assays (Promega, Manheim, Germany), according to the manufacturer's protocol. All experiments were carried out in triplicate.

Primary antibodies used in this study were mouse anti-human monoclonal SIX1 antibody (1:1,000 dilution, sc-514441; Santa Cruz Biotechnology, CA, USA) and mouse anti-human monoclonal GADPH antibody (1:1000 dilution, sc-365062; Santa Cruz Biotechnology). Total protein was extracted from tissues, cell lines, and transfected cells using ice-cold RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, Haimen, China), and subjected to Bicinchoninic Acid Protein assay kit (BCA; Thermo Fisher Scientific, Inc., Rockford, IL, USA) to measure its concentration. Equal proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes (Millipore, Bedford, MA, USA) and then blocked in 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated with specific primary antibodies at 4°C overnight, followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. The specific signals were visualized by using ECL chemiluminescent kit (Pierce Biotechnology, Inc., Rockford, IL, USA).

Data are presented as mean ± SD, and compared using SPSS version 13.0 software (Chicago, IL, USA). P-value <0.05 was considered statistically significant.

In an attempt to explore the roles of miR-362 in cervical cancer, we first measured its expression in cervical cancer tissues and matched adjacent normal tissues using qRT-PCR. The expression levels of miR-362 were obviously reduced in cervical cancer tissues compared with those in matched adjacent normal tissues (Fig. 1A, P<0.05). We also detected miR-362 expression in cervical cancer cell lines. In comparison with an immortalized HPV-negative skin keratinocyte line HaCaT, miR-362 was significantly downregulated in cervical cancer cell lines HeLa, CaSki, C33A and SiHa (Fig. 1B, P<0.05). These results suggested that miR-362 may be involved in the regulation of malignant properties of cervical cancer.

We further evaluated the association between miR-362 expression and clinicopathological features in cervical cancer. As shown in Table II, miR-362 expression was significantly correlated with FIGO stage (P=0.010), lymph node metastasis (P=0.034) and vascular invasion (P=0.039). However, there were no significant correlations between miR-362 expression and age (P=0.284), tumor size (P=0.315) or histological grades (P=0.439).

To further investigate the roles of miR-362 in cervical cancer, miR-362 mimics were injected into HeLa and CaSki cells which expressed relatively lower miR-362 expression. After transfection at 48 h, qRT-PCR was performed to evaluate the transfection efficiency. As shown in Fig. 2A, miR-362 expression was markedly increased in miR-362 mimic-transfected HeLa and CaSki cells as compared with NC groups (P<0.05).

MTT assay was used to investigate the effect of miR-362 overexpression on cervical cancer cell proliferation. As presented in Fig. 2B, miR-362 mimics obviously suppressed the HeLa and CaSki cells proliferation compared with NC (P<0.05). Cell migration and invasion assay was adopted to evaluate the effect of miR-362 on cervical cancer metastasis. As shown in Fig. 2C, ectopic of miR-362 expression impaired the migration and invasion of both HeLa and CaSki cells (P<0.05). Collectively, miR-362 acted as a tumor suppressor on malignant proliferation, migration and invasion in cervical cancer cells, consequently it is involved in the progression of cervical cancer.

Based on bioinformatics analysis with TargetScan and miRanda, SIX1 was identified as a potential target of miR-362 (Fig. 3A). To further explore whether SIX1 was the direct target of miR-362, luciferase reporter assay was used. As shown in Fig. 3B, miR-362 significantly decreased the luciferase activities of PGL3-SIX1-3′UTR Wt (P<0.05) but not the PGL3-SIX1-3′UTR Mut in the HEK293T cells, suggesting direct regulation of miR-362 in the 3′UTR of SIX1.

To determine the regulation effects of miR-362 on SIX1 expression, qRT-PCR and western blot analysis were performed in HeLa and CaSki cells after transfection with miR-362 mimics or NC. The results showed that miR-362 overexpression reduced SIX1 mRNA (Fig. 3C, P<0.05) and protein (Fig. 3D, P<0.05) expression in HeLa and CaSki cells. These results suggested that SIX1 was a direct target of miR-362.

Next, we measured SIX1 endogenous expression in cervical cancer tissues and matched adjacent normal tissues. The results of qRT-PCR and western blot analysis showed that SIX1 mRNA (Fig. 4A, P<0.05) and protein (Fig. 4B, P<0.05) was both significantly upregulated in cervical cancer tissues compared with matched adjacent normal tissues. In additon, the expression level of SIX1 in cervical cancer cell lines was also determined, and revealed that SIX1 protein level was higher in cervical cancer cell lines than that in HaCaT (Fig. 4C, P<0.05). Moreover, Spearman's correlation analysis was performed to explore the association between miR-362 expression and SIX1 mRNA in cervical cancer tissues. As shown in Fig. 4D, SIX1 mRNA expression was inversely correlated with miR-362 expression in cervical cancer tissues (r=−0.7179, P<0.001).

In order to shed light on the roles of SIX1 in cervical cancer, MTT assay, cell migration and invasion assay were carried out in HeLa and CaSki cells transfected with SIX1 siRNA or NC siRNA. After transfection for 72 h, western blot analysis was performed to determine the SIX1 expression. As shown in Fig. 5A, SIX1 siRNA caused a significantly repression on SIX1 protein expression levels (P<0.05). Functional assays revealed that downregulation of SIX1 decreased the proliferation (Fig. 5B, P<0.05), migration and invasion (Fig. 5C, P<0.05) in HeLa and CaSki cells. These results indicated that SIX1 knockdown could simulate the roles of miR-362 overexpression on cell proliferation, migration and invasion of cervical cancer, suggesting SIX1 was a functional downstream target of miR-362.

To confirm that miR-362 inhibited proliferation, migration and invasion by directly targeting SIX1, rescue experiments were performed. pcDNA3.1-SIX1 or pcDNA3.1 was transfected into HeLa and CaSki cells. The transfection efficiency was assessed using western blot analysis, and found that SIX1 protein was significantly upregulated in pcDNA3.1-SIX1 transfected HeLa and CaSki cells (Fig. 6A, P<0.05).

Rescue experiments indicated that restoration of SIX1 was sufficient to abolish proliferation (Fig. 6B, P<0.05), migration and invasion (Fig. 6C, P<0.05) induced by miR-362 overexpression in HeLa and CaSki cells. These results suggested that the inhibition of proliferation, migration and invasion of cervical cancer induced by miR-362 overexpression was at least partly through repression of the SIX expression.