A total of 50 OC tissues and adjacent non-tumor tissues were obtained from patients who underwent surgical resection between January 2004 and December 2008 in the Department of Gynecology, Cangzhou Central Hospital (Cangzhou, China). The age range was 18–75 years with a median age of 50.6 years. Stage was retrospectively assessed for every patient based on a modified International Federation of Gynecology and Obstetrics (FIGO) staging system (20). No patients received chemotherapy before surgery. After written informed consent was obtained from every patient, the clinical samples were used for this study and were stored in liquid nitrogen. The protocol involving patients' samples in this study was approved by the Institutional Research Ethics Committee of Cangzhou Central Hospital. The clinicopathological characteristics of all enrolled patients were presented in Table I.

The present study also used 5 human OC cell lines (CAOV3, SKOV-3, ES-2, HO-8910 and OVCAR3) and the immortalized human fallopian tube FTE187 epithelial cell line cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The OC cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). FTE187 cells were kept in Medium 199 and MCDB105 medium (Sigma-Aldrich; Merck KGaA) containing 10% FBS and 10 ng/ml epidermal growth factor (Sigma-Aldrich; Merck KGaA). All cells were maintained at 5% CO₂ in a humidified atmosphere of 37°C.

The expression vector of miR-19b (pCMV-miR-19b) and the corresponding control (pCMV-MIR), synthetic oligonucleotide against miR-19b (miR-19b inhibitors) and control oligonucleotide (negative control) were obtained from OriGene Technologies, Inc. (Rockville, MD, USA). miR-19b expression vectors (400 ng) and miR-19b inhibitors (100 nM) were transfected into SKOV-3 and OVCAR-3 cells, respectively, using Lipofectamine 2000 following the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent experiments were performed 48 h after transfection.

Isolation of the miRNA fraction from OC tissues and cells was performed using the Isolation of Small RNA kit (Macherey-Nagel GmbH, Düren, Germany). For miRNA analysis, RNA was reverse-transcribed using a TaqMan™ MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using the Kapa Probe Fast qPCR Master Mix (Kapa Biosystems, Inc., Wilmington, MA, USA). The thermocycling conditions were as follows: Incubation at 95°C for 60 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, as previously described (21). The primers used were as follows: Has-miR-19b (RT primer: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGTT-3′; forward, 5′-TGTGCAAATCCATGCAAAACTGA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′), U6 forward, 5′-TCGGCAGCACATATACTAA-3′ and reverse, 5′-ATGGAACGCTTCACGAAT−3′. The relative expression of miR-19b was shown as fold difference relative to U6 using the 2^−ΔΔCq method (22).

Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was used to obtain cellular proteins, followed by quantification with a Bradford Protein assay kit (Beyotime Institute of Biotechnology). In total, 20–40 µg protein per lane was subjected to 10% SDS-PAGE and were transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, then were incubated with primary antibodies against the following proteins: PTEN (cat no. sc-7974; 1:1,500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), RAC serine/threonine-protein kinase (AKT; cat no. 9272; 1:1,500), phosphorylated (p)-AKT (cat no. 4060; 1:500; both Cell Signaling Technologies, Inc., Danvers, MA, USA) and GAPDH (cat no. sc-47724; 1:1,500; Santa Cruz Biotechnology, Inc.) overnight at 4°C, and were then incubated with horseradish peroxidase-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies for 1 h at room temperature (cat nos. 7074 and 7076; 1:2,000; Cell Signaling Technologies, Inc.). The proteins were visualized using the Super Signal West Femto kit (Thermo Fisher Scientific, Inc.) and the signal was detected using the Bio-Rad Gel Imaging system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Image J software (version 1.41; National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein levels.

For the wound-healing assay, OC cells transfected with different vectors were seeded into 6-well plates and cultured to confluence. Wounds were then made using a 100-µl pipette tip and the cells were incubated for 12 h. The width of the wound was visualized using phase-contrast microscopy (magnification, ×100) at 0 and 12 h after the wound was made.

The migratory and invasive ability of OC cells were evaluated using Transwell assays. SKOV-3 and OVCAR-3 cells (1×10⁴) re-suspended in DMEM were seeded into the upper chamber of the Transwell inserts of 8-µm with (EMD Millipore, Billerica, MA, USA). The lower chamber contained 800 µl DMEM supplemented with 20% FBS. The upper chamber was coated with Matrigel when the invasion assays were performed, but not when the migration assay was performed. At 24 h after the cells were seeded, OC cells on the lower surface were fixed with 4% paraformaldehyde (10 min at room temperature) and stained with 0.1% crystal violet (20 min at room temperature), and the number of migrated and invaded cells were then counted in 5 fields under a light microscope (magnification, ×200).

OC cells were seeded in 6-well plates 1 day prior to transfection. These cells were transfected with a PTEN-3′-UTR vector (Promega Corporation, Madison, WI, USA) along with miR-19b mimic, miR-19b inhibitor or mutant miR-19b vector, and pRL-SV40 Renilla plasmid (Promega Corporation) using Lipofectamine 2000. At 48 h after transfection, the Dual-Luciferase Reporter Assay system (Promega Corporation) was used to measure the relative firefly and Renilla (normalization) luciferase activities of OC cells.

Data are presented as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, Inc., USA). P<0.05 was considered to indicate a statistically significant difference. Comparisons between two groups were performed using unpaired Student's t-test; comparisons between multiple groups were performed using one-way analysis of variance with post hoc Tukey's test.

The expression level of miR-216a in tissue samples from 50 patients with OC and matched non-tumor tissues was examined by RT-qPCR. The results of RT-qPCR analysis demonstrated that miR-19b was significantly overexpressed in OC tissues samples compared with the matched non-tumor tissues (P<0.05; Fig. 1A). The expression level of miR-19b in five OC cell lines and FTE187 cells was also examined. Compared with that in FTE187 cells, the expression of miR-19b in each of the five OC cell lines was significantly increased (P<0.05; Fig. 1B). Among OC cells lines, the expression level of miR-19b was highest in OVCAR-3 cells and was lowest in CAOV3 cells.

Next, whether the miR-19b expression level was associated with the clinicopathological features of OC patients was investigated. As shown in Table I, patients with high expression level of miR-19b had a significantly increased percentage to be at an advanced FIGO stage (P=0.018). Furthermore, increased miR-19b expression levels were significantly associated with the lymphatic metastasis of OC patients (P=0.047). These data indicated that an elevated miR-216a level is a promising biomarker for evaluating disease progression for OC patients.

Following the observation of the significantly elevated level of miR-19b in OC samples and cell lines, the biological function of miR-19b was examined in OC cells. A miR-19b mimic and miR-19b inhibitor were transfected to increase and inhibit the miR-19b expression level, respectively, in SKOV-3 and OVCAR-3 cells. The migratory and invasive behavior of OC cells were also examined following alteration the expression of miR-19b in OC cells. First, transfection with the miR-19b inhibitor significantly reduced the expression level of miR-19b in OVCAR-3 cells (P<0.05; Fig. 2A). The results of the wound-healing assay revealed that the migration of OVCAR-3 cells was significantly inhibited following inhibition of miR-19b expression (P<0.05; Fig. 2B). Furthermore, the results of the Transwell assay demonstrated that the migration and invasion of OVCAR-3 cells were significantly inhibited following downregulation of miR-19b (P<0.05; Fig. 2C).

Transfection of the miR-19b expression vector into CAOV-3 cells significantly increased the expression level of miR-19b (P<0.05; Fig. 3A). Subsequently, the migration of CAOV-3 cells was significantly increased (P<0.05; Fig. 3B and C), as was the invasion of CAOV-3 cells (P<0.05; Fig. 3C). These data indicated that miR-19b could increase the migration and invasion of OC cells.

Next the underlying mechanisms responsible for the functional influence of miR-19b on OC cells were examined. Numerous studies confirmed that PTEN and downstream AKT signaling served a notable role in regulating the metastasis of OC cells (23–25). Therefore, whether miR-19b could modulate the expression of PTEN and affect the PTEN/AKT pathway was examined. The results of western blot analysis revealed that overexpression of miR-19b significantly reduced the expression of PTEN in CAOV-3 cells (P<0.05; Fig. 4A). The phosphorylation of AKT, which was under the control of PTEN (26), was significantly increased (P<0.05; Fig. 4A). Inhibition of miR-19b in OVCAR-3 cells significantly increased the expression of PTEN (P<0.05; Fig. 4B) and decreased the phosphorylation of AKT (P<0.05; Fig. 4B). These results indicated that miR-19b could directly regulate the activity of the PTEN/AKT signaling pathway.

Following confirmation of the fact that miR-19b could inhibit the expression of PTEN, whether miR-19b could modulate the expression of PTEN by interacting with the 3′-UTR of PTEN was investigated. As shown in Fig. 5A, the 3′-UTR of PTEN contained the complementary sequence for the seed region of miR-19b, indicating that miR-19b could potentially interact with the 3′-UTR of PTEN. A luciferase assay was performed to confirm the interaction between miR-19b and the 3′-UTR of PTEN. Overexpression of miR-19b significantly decreased the luciferase activity of PTEN 3′-UTR (P<0.05; Fig. 5B), whereas inhibition of miR-19b significantly reduced the lucifease activity of PTEN 3′-UTR (P<0.05; Fig. 5B). Next, the mutant miR-19b was transfected into OC cells, which did not affect the luciferase activity of the PTEN 3′-UTR reporter (Fig. 5B). Taken together, these data indicated miR-19b could modulate the expression of PTEN by directly interacting with the 3′-UTR of PTEN.