Human ovarian epithelial adenocarcinoma SKOV3 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and endometrioid-type ovarian epithelial carcinoma 59M cells were purchased from the European Collection of Authenticated Cell Cultures; Public Health England (Salisbury, UK). SKOV3 cells were cultured in McCoy's 5A medium (Thermo Fisher Scientific, Inc., Waltham, MA) and 59M cells in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Lonza Group, Basel, Switzerland), 2 mM L-glutamine, 100 µg/ml streptomycin and 100 units/ml penicillin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

The sequence of the OGT-targeting small interfering RNA (siRNA) used was 5′-GGATGCTTATATCAATTTAGG-3′. Negative control siRNA oligonucleotides (F: 5′-CCGGTACGTGACACGTTCGGAGAATTCTCGAGAATTCTCCGAACGTGTCACGTTTTTTG-3′; R: 5′-AATTCAAAAAACGTGACACGTTCGGAGAATTCTCGAGAATTCTCCGAACGTGTCACGTA-3′) were purchased from Eurogentec (Liège, Belgium). siRNA (1.6 µg) was diluted with Opti-MEM I Reduced Serum Medium (Invitrogen; Thermo Fisher Scientific, Inc.) to a final volume of 100 µl. DreamFect reagent (8 µl; OZ Biosciences, Marseille, France) was diluted with Opti-MEM I Reduced Serum Medium to a final volume of 100 µl. The RNAi solution and transfection reagent were then combined and incubated for 20 min at room temperature. The 200 µl mixture was then added to 80% confluent cells (1.2×10⁶ cells per well) maintained in 6-well plates, with 1.8 ml of culture medium per well. Cells were transfected onceover 24 h for 4 days. OGT expression and activity were detected at 96 h by western blot analysis, and transfected cell invasion and migratory capacity were evaluated by Transwell assay.

The sequence of the RhoA-targeting siRNA was 5′-AAGCAGATGAGAATGACGTCGGTG-3′, and negative control siRNA (5′-ACGTGACACGTTCGGAGAATT-3′) was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). siRNAs were transfected into SKOV3 and 59M cells by electroporation with the Amaxa Nucleofector (Lonza Group) according to the manufacturer's protocols, then lysed 24 h later and analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis to measure RhoA mRNA and protein expression levels, respectively.

Total RNA in cells was extracted using a total RNA isolation kit (A&A Biotechnology, Gdynia, Poland). cDNA was obtained by reverse transcription of 1 µg of total RNA in a 20 µl reaction using a RevertAid™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) and was amplified using a TaqMan^® Gene Expression Assay (Applied Biosystems; Thermo Fisher Scientific, Inc.), using primers specific for the target proteins. The sequences of the primers were as follows: 5′-CGGGAGCTAGCCAAGATGAAG-3′ (F) and 5′-GCTTGCAGAGCAGCTCTCGTA-3′ (R) for RhoA. 5′-GGCCGTGAAGTCGTCAGAAC-3′ (F) and 5′-GCCACGATGCCCAGGAA-3′ (R) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The two genes were amplified by a first step of 120 sec at 95°C, followed by 45 cycles of 30 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C. mRNA expression of RhoA was calculated using the formula 2^−ΔΔCq (35) and was normalized to GAPDH expression levels. mRNA expression levels in cells transfected with RhoA siRNA are presented relative to mRNA expression levels in cells with negative control siRNA.

Cell migration was evaluated by Transwell assay using Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA) (36). A total of 600 µl cell culture medium, with or without 5 µM Thiamet-G (an OGN inhibitor) or 50 µM Y-27632 (a ROCK inhibitor) was added in the lower chamber. Culture medium (100 µl) containing 1×10⁵ SKOV3 or 59M cells and 1% FBS, was plated into the upper chamber, with or without Thiamet-G or Y-27632. The cells on the undersurface of the upper chamber were stained with crystal violet 20 h later and were observed using a light microscope. A total of six fields at ×100 magnification were selected at random to measure the average cell coverage using ImageJ software version 1.45s (National Institutes of Health, Bethesda, MD, USA). Invasion assays were performed using the same protocol as the migration assay, but the upper face of the polycarbonate membrane in the upper chamber was covered with 1 mg/ml Matrigel (BD Biosciences) and the invasive cells were detected following 24 h incubation.

Cells were lysed for 15 min at ice bath using a lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 1 mM MgCl₂, 150 mM NaCl, 1 mM Na₃VO₄, 50 mM NaF, 1.5 mM EDTA, 10% glycerol, 20 mM β-glycerophosphate, 10 µg/ml aprotonin, 1 µM pepstatin A) containing 5 µM PUGNAc (an OGA inhibitor; Toronto Research Chemicals, Inc., North York, Canada). Protein samples (50 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% non-fat dried milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. Antibodies specific to O-GlcNAcylation (RL2; 1:1,000; Affinity BioReagents, Golden, CO, USA) and OGT (F-12; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used. RhoA (67B9; 1:1,000), MLC (3672; 1:1,000) and phosphorylated (p-)MLC (3671; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH antibody (sc-25778; 1:2,000) and horseradish peroxidase-linked goat anti-mouse (sc-2005; 1:2,000) and goat anti-rabbit (sc-2004; 1:2,000) IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Development was carried out using an enhanced chemiluminescence western blotting detection reagent (GE Healthcare Life Sciences, Chalfont, UK).

RhoA activity was analyzed using Rhotekin Rho binding domain (Upstate Biotechnology; Thermo Fisher Scientific, Inc.) bound to glutathione agarose beads to pulldown the active GTP-bound RhoA form from ovarian cell lysates in lysis buffer (20 mM HEPES, pH 7.5, 0.5% NP-40, 100 mM NaCl, 0.2% deoxycholic acid, 10% glycerol, and 10 mM MgCl₂) supplemented with protease and phosphatase inhibitors (37). GTP-bound RhoA and total RhoA were evaluated by western blot analysis as above, using RhoA antibody (67B9; 1:1,000; Cell Signaling Technology, Inc.).

Cells were starved in serum-free medium for 24 h, then incubated at 37°C for 60 min with or without Thiamet-G at 5 µM concentration. The cells were subsequently lysed for 15 min at 4°Cin cell lysis buffer [100 mM NaCl, 1 mM Na₃VO₄, 40 mM Na₄P₂O₇, 20 mM NaF, 30 mM HEPES/NaOH (pH 7.4), 1% Triton X-100, 1 mM EGTA, 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin and 10 µg/ml aprotinin], and cell lysates were centrifuged for 10 min at 4°C. The cell extracts were then used for western blot analysis using MLC and p-MLC antibodies, as above.

All experiments were repeated at least three times. SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA) was used for analysis, and data were expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

Two human ovarian cancer cell lines, SKOV3 and 59M, were used to determine the involvement of O-GlcNAcylation in ovarian cancer. To alter the O-GlcNAcylation level, cells were transfected with OGT-targeting siRNA or treated with the OGA inhibitor Thiamet-G (5 µM) for 24 h. Western blot analysis revealed that OGT silencing decreased the level of global O-GlcNAc in SKOV3 (Fig. 1A) and 59M cells (Fig. 1B) compared with cells transfected with control siRNA. OGT protein expression levels were also visibly reduced in SKOV3 (Fig. 1A) and 59M cells (Fig. 1B) transfected with OGT siRNA compared with cells transfected with control siRNA. Thiamet-G treatment visibly increased global O-GlcNAc levels in SKOV3 (Fig. 1A) and 59M cells (Fig. 1B) compared with untreated control cells, indicating that it effectively inhibited OGA activity.

The effect of O-GlcNAcylation on ovarian cancer malignancy was investigated via in vitro analysis of cell migration and invasion using a Transwell assay. Transfection with OGT siRNA significantly decreased migration (SKOV3 cells, P=0.007; 59M cells, P=0.009; Fig. 2A) and invasion (SKOV3 cells, P=0.006; 59M cells, P=0.008; Fig. 2B) in OGT siRNA transfected cells compared with control siRNA transfected cells. However, Thiamet-G treatment significantly increased migration (SKOV3 cells, P=0.007; 59M cells, P=0.009; Fig. 2A) and invasion (SKOV3 cells, P=0.007; 59M cells, P=0.006; Fig. 2B) in treated cells compared with untreated controls. This indicates that a positive correlation exists between the intracellular global O-GlcNAcylation level and the motility of ovarian cancer cells.

It has previously been reported (22–27) that Rho GTPases are associated with cell motility, with RhoA stimulating ROCK and MLC to regulate these cellular events. To determine how O-GlcNAcylation modulates ovarian cancer cell motility, RhoA activity was detected by pull-down assay. The results revealed that Thiamet-G treatment-induced O-GlcNAcylation upregulation visibly enhanced RhoA activity at 3 and 6 h in SKOV3 and 59M cells compared with untreated control cells (Fig. 3A), while downregulation of O-GlcNAcylation induced by OGT silencing visibly reduced RhoA activity in SKOV3 and 59M cells compared with control cells (Fig. 3A). MLC phosphorylation is stimulated by RhoA through ROCK activation (25), so MLC phosphorylation was analyzed by western blotting. The results indicated that O-GlcNAcylation upregulation increased MLC phosphorylation in SKOV3 and 59M cells compared with untreated controls (Fig. 3B), and O-GlcNAcylation downregulation attenuated this phosphorylation in SKOV3 and 59M cells compared with control cells (Fig. 3B). This suggests that the RhoA/ROCK/MLC signal pathway may be closely associated with O-GlcNAcylation and the regulation of motility in ovarian cancer cells.

To determine whether O-GlcNAcylation affected ovarian cancer cell motility by targeting RhoA/ROCK signaling, RhoA was knocked down by RNAi and interference efficiency was assessed using RT-qPCR and western blot analysis to measure mRNA and protein expression levels, respectively. RhoA mRNA and protein expression levels were effectively decreased in SKOV3 cells transfected with RhoA siRNA compared with control cells (Fig. 4A and B, respectively). RhoA silenced and non-silenced cells were subsequently treated with or without Thiamet-G, and cell migration and invasion were evaluated by Transwell assay. Thiamet-G treatment resulted in a significant increase in migration and invasion compared with control cells in SKOV3 (P=0.005 and P=0.006, respectively; Fig. 4C and D, respectively) and 59M cells (P=0.009 and P=0.005, respectively; Fig. 4C and D, respectively). RhoA silencing significantly attenuated cell migration and invasion in SKOV3 (P=0.004 and P=0.006, respectively; Fig. 4C and D, respectively) and 59M cells (P=0.007 and P=0.004, respectively; Fig. 4C and D, respectively) compared with control cells. No significant difference was observed in migration or invasion between RhoA silenced cells and RhoA silenced cells treated with Thiamet-G (Fig. 4C and D, respectively). These findings suggest that RhoA is involved in the regulation of O-GlcNAcylation in ovarian cancer cell motility.

Y-27632 is able to effectively inhibit ROCK activity (38) and is often used in the investigation of the ROCK signal pathways (39). Therefore, Thiamet-G treated and untreated SKOV3 and 59M cells were treated with or without 50 µM Y-27632, and cell motility was analyzed. Thiamet-G treatment resulted in a significant increase in migration and invasion compared with control cells in SKOV3 (P=0.005 and P=0.008, respectively; Fig. 5) and 59M cells (P=0.007 and P=0.003, respectively; Fig. 5). Y-27632 treatment significantly inhibited cell migration and invasion in SKOV3 (P=0.006 and P=0.004, respectively; Fig. 5) and 59M cells (P=0.007 and P=0.003, respectively; Fig. 5) compared with control cells. No significant difference was observed in invasion or migration between Y-27632 treated and Thiamiet-G+Y-27632 treated cells (Fig. 5). These results suggest that O-GlcNAcylation regulates ovarian cancer cell motility through the RhoA/ROCK signal pathway.