A total of 126 fresh-frozen human normal and malignant ovarian tissues were collected at the Department of Obstetrics and Gynecology of the First Affiliated Hospital of Zhengzhou University. Both tumor and normal ovarian tissues were histologically examined, and the tumor histology was serous cystadenocarcinoma. The tissue collection used for quantitative real-time PCR analysis included 30 normal ovarian tissue sections and 96 serous ovarian carcinoma tissues. Formalin-fixed, paraffin-embedded tissues, matching the frozen cases, were retrieved for immunohistochemical analysis. The patients had not received any local or systemic anticancer treatments prior to the surgery. The relevant clinical characteristics of the studied subjects are shown in Table I.

Informed consent was obtained from each patient, and the study was approved by the Institute Research Ethics Committee at the Cancer Center.

The ovarian cancer cell lines OVCAR-3 and SK-OV-3 were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cell lines were maintained in RMPI-1640 supplemented with 10% fetal bovine serum (FBS) (both from HyClone, USA), 100 U/ml of penicillin, and 100 g/ml of streptomycin at 37°C in 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC) and cultured in medium 199 (Gibco-Invitrogen, USA), supplemented with 20% FBS, 50 mg/ml endothelial cell growth supplement (ECGS) (BD Biosciences, USA), 100 mg/ml heparin (Sigma, USA) and 1% penicilin-streptomycin. Cells were all cultured at 37°C in a 5% CO₂ incubator.

The human miR-497 precursor sequences were cloned into the lentivirus-based expression plasmid pLenti-6.3 (Invitrogen, USA). Viral packaging and infection were performed according to the standard protocols as recommended by the manufacturer. Cells were infected with 1×10⁷ lentivirus transducing units in the presence of 10 μg/ml Polybrene (Sigma-Aldrich, St. Louis, MO, USA). An empty lentiviral vector was used as the negative control. The sequence-specific miR-497 inhibitor (anti-miR-497) and its control (anti-NC) were from BioCat GmbH (Heidelberg, Germany).

Total RNA was extracted using TRIzol reagent (Invitrogen). To quantitate miR-497 expression, total RNA was polyadenylated and reversely transcribed using NCode miRNA First-Strand cDNA synthesis kit (Invitrogen). To measure the mRNA levels of VEGFA, total RNA was reversely transcribed using the PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan). Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (Takara) in Agilent Mx3005P. Primers used for miR-497 were: 5′-GTGCAGGGTCCGAGGT-3′ (forward) and 5′-TAGCCTGCACACTGTGGT-3′ (reverse). Primers used for VEGFA were: 5′-GTTTGACAAGACCACCAAACT-3′ (forward) and 5′-CCGCATAATCTGCATGGTGAT-3′ (reverse). GAPDH was used as an internal control. All samples were normalized to internal controls, and fold-changes were calculated through relative quantification (2^−ΔΔCt).

Double-stranded oligonucleotides corresponding to the wild-type (WT, 3′-UTR) or mutant (Mut, 3′-UTR) miR-497 binding site in the 3′-UTR of VEGFA were synthesized and subcloned into the GV272 reporter vector (GV272: SV40-Luciferase-MCS-PolyA was purchased from Shanghai GeneChem Co., Ltd.). Cells were transfected with the appropriate plasmid and miR-497 duplex. Luciferase assays were carried out using the Luciferase reporter assay system (Promega, USA) 48 h after transfection. Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity.

Transfected cells were cultured in serum-free medium (SFM) for endothelial cells for 12 h. The TCM was centrifuged sequentially at 500 × g to remove the detached cells and at 12,000 × g to discard cell debris (4°C, 10 min each). Aliquots of TCM were stored at −80°C until used.

HUVECs (2.5×10⁴) were grown in the absence or presence of 100% TCM for 10 h at 37°C in a 96-well plate coated with Matrigel (3432-005-01; R&D Systems, Minneapolis, MN, USA). The formation of capillary-like structures was captured under a light microscope. The branch points of the formed tubes, which represent the degree of angiogenesis in vitro, were scanned and quantitated in five low-power fields.

For each sample, total protein extracts were separated on SDS-PAGE gels and transferred to nitrocellulose membranes, which were blocked with 5% non-fat dry milk for 2 h and incubated with antibodies. The antibodies for VEGFA, VEGFR2 and ERK1/ERK2 were obtained from LifeSpan BioSciences Inc. The antibodies for FAK and SHP-1 were obtained from Cell Signaling Technology. The corresponding bands were quantified by image processing software, ImageJ (NIH, http://rsb.nih.gov/ij/).

The antibodies included rabbit anti-VEGFA/VEGF polyclonal antibody (LifeSpan BioSciences, Inc.) and rabbit anti-CD34 polyclonal antibody (ZSGB-Biotechnology, China). Immunodetection was performed with a Vectastain ABC kit (Vector Laboratories). The microvessel density (MVD) in tumor tissues, which represents the degree of angiogenesis in vivo, was evaluated by staining for CD34, an endothelial cell marker. Any discrete cluster or single cell stained for CD34 was counted as one microvessel.

The data are expressed as the means ± standard error of the mean (SEM) from at least three independent experiments. The values for the capillary tube formation and luciferase activity assays are from three independent experiments that were performed in duplicate. The differences between the groups were analyzed by the Student’s t-test when two groups were compared or by one-way ANOVA when more than two groups were compared. Analyses were performed with GraphPad Prism, version 5 (GraphPad Software, Inc., San Diego, CA, USA). Correlations between two variables were explored with the Spearman’s correlation coefficient. All statistical tests were two-sided, and p<0.05 was considered to indicate a statistically significant result.

To assess the clinical importance of miR-497 in ovarian cancer, we examined miR-497 levels in ovarian cancer tissues and normal ovarian tissues by real-time PCR. As shown in Fig. 1A, the expression levels of miR-497 in the ovarian cancer tissues were much lower than those in the normal ovarian tissues (p=0.0047). Furthermore, lower miR-497 levels were associated with higher MVDs (p=0.0032; Fig. 1B and C), suggesting that miR-497 downregulation may contribute to ovarian cancer progression by promoting tumor angiogenesis.

miR-497 inhibits ovarian cancer cell angiogenesis. To explore the effect of miR-497 on ovarian cancer angiogenesis, an in vitro capillary tube formation assay was carried out in SK-OV-3 and OVCAR-3 cells. We found that, compared with the control media (SFM), TCM from the negative control (NC)-transfected and non-transfected cells promoted the ability of HUVECs to develop more capillary-like structures (Fig. 2A and B). Tube formation was reduced dramatically in the HUVECs that were grown in TCM from the miR-497-transfectants, to a level comparable to that of the HUVECs cultured in SFM (Fig. 2A and B). In contrast, the suppression of endogenous miR-497 in ovarian cancer cells resulted in enhanced capillary tube formation (Fig. 2C and D).

We further investigated the molecular mechanism by which miR-497 inhibits ovarian cancer angiogenesis. Putative targets of miR-497 were predicted with TargetScan. Among these, VEGFA was selected for further research due to its well-known importance in tumor angiogenesis. Dual-luciferase reporter assay revealed that the co-transfection of miR-497 significantly inhibited the activity of firefly luciferase reporter with wild-type 3′UTR of VEGFA, whereas this effect was abrogated when the predicted 3′UTR binding site was mutated (Fig. 3A and B). Western blot analysis showed that the ectopic expression of miR-497 in the SK-OV-3 and OVCAR-3 cells resulted in the downregulation of VEGFA at the protein level (Fig. 3C and E). To confirm the effects of miR-497 on VEGFA, we transferred anti-miR-497 into ovarian cancer cells and found that inhibition of miR-497 increased the protein level of VEGFA (Fig. 3D and F). Moreover, an inverse correlation between miR-497 and VEGFA expression was confirmed in the human ovarian cancer tissues (Fig. 3G and H). These data indicate that miR-497 may negatively regulate VEGFA expression by directly targeting its 3′UTR.

We further validated that VEGFA mediates the anti-angiogenic function of miR-497. Suppression of VEGFA in ovarian cancer cells significantly reduced capillary tube formation (Fig. 4A and B). In contrast, overexpression of VEGFA in the miR-497-transfected cells attenuated the anti-angiogenic effects of miR-497 (Fig. 4D). In addition, human ovarian cancer tissues with higher MVD exhibited higher levels of VEGFA (p=0.0060, Fig. 4C), corresponding to the correlation between lower miR-497 expression and higher MVD/VEGFA levels in ovarian cancer tissues (Figs. 1B and 3G).

VEGFR2 is the major receptor of VEGFA that mediates angiogenic activity, and the VEGF/VEGFR2 signaling pathway plays a crucial role in angiogenesis (21). To ascertain whether miR-497 affects expression of VEGFA through this signaling pathway, we determined the effects of miR-497 on the activation of VEGFR2 by immunoblotting of phospho-VEGFR2. As shown in Fig. 4, the miR-497-transfected ovarian cancer cells displayed significantly decreased phosphorylation of VEGFR2 when compared with the negative control cells.

To further understand how miR-497 exerts its anti-angiogenic effects via VEGFR2-mediated signaling, several key VEGFR2 downstream signaling molecules were studied. Western blot assay revealed that compared to the negative control cells, the miR-497-transfected ovarian cancer cells had significantly decreased phosphorylation of AKT and ERK1/2, while the phosphorylation status of other kinases such as SHP-1 and FAK were not significantly affected (Fig. 4E). In contrast, anti-miR-497-transfectants enhanced PI3K/AKT and MAPK/ERK signaling in the ovarian cancer cells (Fig. 4E). These results suggest that miR-497 may inhibit angiogenesis by downregulation of VEGFA expression via the VEGFR2-mediated PI3K/AKT and MAPK/ERK pathways in ovarian cancer cells.