Blockade of ONECUT2 expression in ovarian cancer inhibited tumor cell proliferation, migration, invasion and angiogenesis

One cut homeobox 2 (ONECUT2 or OC‐2) is a newly discovered transcription factor. Aberrant expression of OC‐2 is closely related to cell proliferation, migration, invasion, and angiogenesis. In this study, we found that OC‐2 expression was upregulated in ovarian adenocarcinoma cells, by Western blot analysis. The results of immunohistochemistry showed that the expression of OC‐2 was also increased in malignant ovarian cancer tissue. In order to explore the role of OC‐2 in the development of ovarian cancer, siRNAs that specifically targets OC‐2 were designed. The siRNA targeting OC‐2 could effectively inhibit the vascular endothelial growth factor A (VEGFA) expression, but silence and overexpression of VEGFA did not affect OC‐2 expression. In addition, OC2‐siRNA could block the proliferation, migration, and invasion, and inhibit epithelial–mesenchymal transition and the AKT/ERK signaling pathway, of human ovarian cancer cells in vitro. In a mouse model of ovarian cancer xenograft tumors, OC2‐siRNA could significantly inhibit tumor cell growth and the tumor inhibition rate reached approximately 73%. The results of immunohistochemistry showed that the densities of microvessels stained with CD31, the expression of OC‐2 and VEGFA were significantly decreased in tumors. These data indicated that OC‐2 was an upstream regulator of VEGFA and silencing OC‐2 could inhibit ovarian cancer angiogenesis and tumor growth.

cell precursors, which can be derived from adjacent vessels or from incorporation of bone marrow-derived endothelial precursor cells. 9 However, tumor neovascularization, consisting only of vascular endothelial cells, has been challenged by vasculogenic mimicry (VM). 10 Tang et al 11 indicated that vasculogenic mimicry was an alternative way to supply adequate blood for ovarian cancer. Research on tumor angiogenesis had progressed slowly until important discoveries of the major molecular drivers of tumor angiogenesis: the VEGF family and its homologous receptors. 12 In addition to VEGF, other important pathways also contribute to tumor angiogenesis. 8 One cut homeobox 2 (ONECUT2 or OC-2) was first reported by Patrick et al 13 as the second member of the ONECUT family, whose prototype was hepatocyte nuclear factor-6, characterized by the presence of a single cut domain and by a specific homeodomain. The expression status of OC-2 could be used for bladder cancer epigenetic analysis and the results had high sensitivity and specificity. 14 As an angiogenic and EMT-related transcription factor, OC-2 plays a key role in tumor cell invasion, metastasis, EMT, and oncogenesis. 15 FBS. All these cells were cultured in an incubator with 95% humidity and 5% CO 2 at 37°C.
Ovarian cancer cells (SKOV3 and COV-504, 2 9 10 5 cells/well) were added to 6-well plates for 24 hours in DMEM with 10% FBS before transfection. SKOV3 and COV-504 cells were transfected with siRNAs or pAAV-VEGFA in ViaFect Transfection Reagent (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. After transfection, the cells were cultured in a 37°C incubator for 24 hours for the further experiments.

| Cell proliferation assay
The cell viability was measured by CCK-8 assay (Dojindo Molecular Technologies

| Western blot analysis
The ovarian cancer cells were washed with cold PBS and lysed in RIPA buffer (Beyotime, Suzhou, China). The lysates were centrifuged at 12 000 g for 8 minutes at 4°C and the total protein in the supernatant were quantified by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA).

| Real-time quantitative PCR analysis
The total RNAs were extracted from cells with TRIzol reagent (Invitrogen, China). The RT reactions were carried out using a SYBR green-containing PCR kit (Qingke, China). The primers for real-time PCR were used to detect the mRNA expression of hypoxia-inducible factor-1a (HIF-1a), VEGFC, hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), Snai1, Snai2, transforming growth factor-b1 (TGF-b1), and Twist1. The sequences of real-time PCR primers are listed in Table 3. The primers were synthesized and purified by Beijing Qingke Biotechnology. All real-time PCR procedures were carried out on the CFX96 detection system (Bio-Rad, USA). 15

| Wound healing assay
SKOV3 and COV-504 cells (4.0 9 10 5 cells/well) transfected with OC2-siRNA were transferred to 6-well plates and cultured in DMEM with 10% FBS. The control groups were transfected with scramble siRNA. After incubation overnight, the cell scratches were made. The plates were washed with serum-free DMEM and the medium was changed to DMEM with 0.5% FBS. After incubation for 24 hours, the migrated cells were imaged with a computerized imaging system.
The cell migration rate of each group was calculated. 19 The cell migration inhibition rate = (1 À OC2-siRNA group migration distance/siRNA NC group migration distance) 9 100%.

| Migration and invasion assays
Migration and invasion assays of ovarian cancer cells were evaluated

| Tube formation assay
The 96-well plates were maintained at 4°C for 2 hours and coated with 60 lL Matrigel matrix per well. The plates were incubated for 30 minutes at 37°C. Then HUVECs (5 9 10 3 cells/well) suspended in M199 medium with Low Serum Growth Supplement (Gibco) were transferred to each well. Conditioned medium (70 lL) of SKOV3 and COV-504 cells transfected with OC2-siRNAs was added in the wells for 6 hours at 37°C in a 5% CO 2 incubator. The tube formation was observed under a microscope (IX71; Olympus, Japan) and the tube numbers were counted in five random high-power fields.  19 At the end of 21 days, nude mice were euthanized and tumors were stripped for immunohistochemistry assays.

| Immunohistochemistry
Immunohistochemistry was carried out as previously described. 19,20 The tumor tissue samples of ovarian cancer patients were provided  Technology), and rabbit anti-mouse ONECUT2 polyclonal antibody (1:500 dilution, ab28466; Abcam). The biotinylated HRP-conjugated secondary antibody was a goat anti-rabbit IgG.

| Statistical analysis
Statistical analyses were carried out by one-way ANOVA followed by the least significant difference test using the statistical software and P < .01 (**) were considered statistically significant.

| Ethics
The hospital ethics committee approved the study protocol, and all patients provided gave consent for the utilization of their tissue samples in this study.

| Overexpression of OC-2 in some ovarian cancer cells and tissues
Previous studies have indicated that OC-2 was expressed highly in liver cancer and colon cancer. 15,16 To explore the expression and sig-

| Silencing OC-2 expression in ovarian cancer cells inhibits endothelial angiogenesis
Anti-angiogenic therapy is used as a primary treatment for ovarian cancer. 21 To test the role of OC-2 in ovarian cancer cells stimulating angiogenesis, the conditioned media were collected from the COV-

| Silencing of OC-2 inhibits phosphorylation of AKT, ERK1/2, and EMT
The pathway of AKT/ERK has been verified to correlate with tumor growth and angiogenesis. [22][23][24] ONECUT-2 decreased the proliferation rate of SKOV3 and COV-504 cells, and showed a time-dependent inhibition of cell growth ( Figure 2D,E). To further assess whether OC-2 affected the downstream signaling pathway involved in cell proliferation and angiogenesis, p-Akt and p-Erk were investigated by Western blot assay. The results showed that silencing OC-2 could inhibit the phosphorylation of Akt and Erk ( Figure 6A).
ONECUT-2 is a transcription factor associated with EMT, and Ncadherin and E-cadherin are important markers of EMT development. 15,25 As a feature of aggressive tumors, EMT is characterized by reduced E-cadherin and increased N-cadherin expression, contributing to a stroma-oriented cellular adhesion profile with increased tumor cell motility and invasive properties. [26][27][28] To explore the relationship between OC-2 and EMT-related markers, N-cadherin and E-cadherin expression were detected after silencing OC-2 by Western blot, and the mRNA expression of Snai1, Snai2, TGF-b1, and Twist1 were detected after silencing OC-2 by real-time PCR.
Our results showed that E-cadherin expression increased and Ncadherin decreased after OC-2 silencing on SKOV3 and COV-504 cells (Figure 6Ba-e). In addition, the real-time PCR results showed that the expression levels of TGF-b1 in COV-504 and SKOV3 cells transfected with siRNA#1 reduced by approximately 56% and 48%, respectively (P < .05). The Snai2 expression levels were downregulated by approximately 45% and 40%, respectively (P < .05). The Twist1 expression levels were decreased by nearly 51% and 53%, respectively (P < .05). In contrast, the Snai1 expression levels declined by 25% and 20%, respectively, but the results did not reach a significant level (Figure 6Bf).
These results implied that OC-2 might play a role through the Akt/Erk signaling pathway to promote ovarian cancer cell proliferation and activate EMT to improve migration and invasion in ovarian cancer cells.

| Silencing OC-2 inhibits ovarian tumor growth and angiogenesis
In the mice were euthanized and the tumors stripped ( Figure 7A). The OC-2 silenced group showed a significant inhibition in tumor growth (P < .05) from the day 7, whereas the siRNA NC group showed a continuous increase ( Figure 7B). In accordance with this observation, the average weight of tumors in the OC-2 silenced group was significantly reduced compared to the control group (P < .01) ( Figure 7C).
These data suggest that OC-2 could be an important regulator of angiogenesis and tumor growth in ovarian cancer.

| DISCUSSION
Previous studies regarding OC-2 focused on the regulatory role of the ONECUT family in the differentiation and growth of liver tissues F I G U R E 7 ONECUT-2 (OC-2) silencing inhibits tumor growth and angiogenesis in an ovarian cancer xenograft nude mice model. A, COV-504 ovarian cancer cells transfected with siRNA#1/scrambled siRNA (siRNA NC) were injected into the right shoulder of BALB/c nude mice (n = 10). Mice were euthanized and the tumors were excised after 21 d. B, Growth curves of tumors. C, Quantitative analysis of tumor weight. D, Paraffin sections of tumors were stained with OC-2, CD31, VEGFA, and HIF-1a antibody. The positive rates were statistically analyzed. E, Tumors were stained with CD31 antibody. Microvessel densities were quantified. The numbers of blood vessels at five high-power fields (9200) per section were counted. F, Positive expression rates of vascular endothelial growth factor A (VEGFA), OC-2, and hypoxia-inducible factor-1a (HIF-1a) in tumors. Data were represented as the means AE SD. *P < .05, **P < .01 and visual cells, as well as the specific diagnosis of bladder cancer. [29][30][31] It was reported that OC-2 existed in the nucleus and its expression was significantly increased in colorectal carcinoma relative to paracancerous normal tissues. 15 Zhang et al 16  These results indicated that high expression of OC-2 in ovarian cancer cells could stimulate cell migration and invasion and break through the epithelial basement membrane. This is particularly relevant to the strong metastatic capacity of highly differentiated malignant ovarian cancer cells. 38 Subsequently, we used siRNA to silence OC-2 expression to investigate its effect on angiogenesis in vitro and in vivo. In vitro, the conditioned medium after silencing OC-2 in ovarian cancer cells significantly inhibited the tube formation numbers of HUVECs. Western blot assay showed that VEGFA expression was decreased after OC-2 silencing, but silencing and overexpression of VEGFA did not significantly affect OC-2 expression. These results indicated that OC-2 was an upstream regulator of VEGFA. In addition, we found that HIF-1a expression was significantly reduced after silencing OC-2 by realtime PCR and the mouse tumor immunohistochemistry experiments.
The mRNA expression levels of VEGFC, HGF, and FGF2 also decreased in varying degrees after silencing OC-2 expression in ovarian cancer cells by RT-PCR. Hypoxia stimulates new blood vessel formation in coronary artery disease, tumor angiogenesis, and diabetic neovascularization. [39][40][41] Hypoxia-inducible factor-1a increase of VEGF expression is due to both transcriptional activation and increased mRNA stability. 42 Tacchini et al 43  naling pathway. We hypothesized that OC-2 might also act through other angiogenic signaling pathways, such as STAT3/GIV, rather than just the Akt/Erk and HIF-1 signaling pathways that we revealed here.
Therefore, silencing OC-2 might affect one or several signaling pathways, inhibiting the expression and secretion of angiogenesis-related factors and ultimately affecting angiogenesis.
In addition, nude mice bearing COV-504 ovarian cancer transfected with OC2-siRNA (siRNA#1) showed significant tumor growth inhibition. The siRNA#1-treated tumors showed a significant reduction in CD31 stained blood vessels, OC-2, VEGFA, and HIF-1a expression compared to the siRNA NC group. These data indicated that inhibition of OC-2 expression could affect tumor growth and angiogenesis.
Our results provided proof of targeting OC-2 for anti-angiogenesis therapy in ovarian cancer; OC-2 might be a marker of ovarian cancer-specific detection.

ACKNOWLEDG MENTS
This work was supported by grants from the State Natural Science

CONF LICT OF I NTEREST
The authors have no conflict of interest.