The human HO8910 epithelial ovarian cancer cell line and human 293FT embryonic kidney cell line were obtained from the Zhongshan University Laboratory Animal Center (Guangzhou, China). HO8910 cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen-Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), while 293FT cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/l glucose (PAA Laboratories, Pasching, Austria), 10% heat-inactivated FBS (Hyclone), 100 U/ml penicillin and 100 µg/ml streptomycin (both from Invitrogen-Life Technologies) in a humidified incubator containing 5% CO₂ at 37°C.

A lentiviral expression vector carrying RhoA shRNA was obtained from GeneCopoeia (Rockville, MD, USA), and the target sequence of RhoA cDNA was 5′-GAAGGCAGAGATATGGCAA-3′. A negative control vector was provided by GeneCopoeia, designated as Lenti-RhoA-NC. Lenti-RhoA-sh and Lenti-RhoA-NC vectors contained enhanced green fluorescent protein (eGFP) cDNA. The recombinant and control lentivirus were then produced via the co-transfection of 293FT cells with the Lenti-Pac™ HIV Expression Packaging kit (GeneCopoeia), and the virus-containing supernatant was harvested at 48 and 72 h post infection. To stably establish the RhoA-knockdown HO8910 subline, cells were grown and infected with lentivirus at a multiplicity of infection of 50, and the infection efficiency was directly measured by the detection of eGFP expression in cells by fluorescence microscopy. The cells were grown in medium containing 1.5 µg/ml puromycin (Invitrogen) for 20 days. After reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis confirmation of RhoA knockdown, RhoA shRNA-lentivirus-infected cells were named as RhoA-KD cells, whereas the control Lenti-RhoA-NC-infected cells were designated as mock cells.

Total cell RNA was isolated from ovarian cancer cells using TRIzol reagent (Invitrogen-Life Technologies) and reverse transcribed into cDNA using the PrimeScript RT-PCR kit (Takara Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. The primers for the human RhoA gene were: forward, 5′-TTCCATCGACAGCCCTGATAGTTTA-3′ and reverse, 5′-CACGTTGGGACAGAAATGCTTG-3′; while GAPDH was used as the internal control with the primers: forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. PCR amplification and real-time fluorescence signaling monitoring was conducted using a Stratagene fluorescence quantitative PCR instrument according to a previous study (20).

Cell lysis was performed using RIPA lysis buffer from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and the protein concentration was measured using the Bradford method with the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). For western blot analysis, equal amounts of protein extracts (30 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto 0.45-µm polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat dry milk solution in Tris-based saline Tween-20 (TBST) for 1 h and then incubated at 4°C overnight with a polyclonal rabbit anti-RhoA antibody at a dilution of 1:100 (no. sc-179; Santa Cruz Biotechnology, Inc.) or a rabbit anti-β-actin antibody at a dilution of 1:1,000 (no. AP0060; Bioworld Technology, Louis Park, MN, USA) in TBST containing 5% non-fat dry milk, followed by incubation with a secondary horseradish peroxidase-conjugated anti-rabbit antibody (no. BS13278; Bioworld Technology) at a dilution of 1:5,000 in TBST containing 5% non-fat dry milk for 1 h at room temperature. The target protein bands visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology) and quantified using Image J 1.44p software (National Institutes of Health, Bethesda, MD, USA).

The altered cell viability after RhoA expression was knocked down in ovarian cancer cells was assayed by the cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously described (21). Stable RhoA-knockdown or control shRNA-infected cells were seeded in 96-well plates at a density of 5×10³ cells/well and grown for up to 5 days. At the end of each experiment, 1 µl of MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added into each well and the plates were incubated for an additional 4-h at 37°C. Then, 100 µl of dimethyl sulfoxide (DMSO) was added into each well to dissolve formazan by agitation for 10 min. The absorbance values (A) of cells were measured at a wavelength of 492 nm using a microplate reader. The experiments were performed in 5 wells and were repeated at least three times. The data were summarized as a percentage of the control using the formula: (OD_RhoA/OD_control) × 100, where OD_RhoA and ODcontrol were the optical densities in RhoA-knockdown and negative control cultures, respectively.

Tumor cell migration and invasion ability were assayed using a 24-well Transwell chamber system with an 8-µm pore size polycarbonate membrane (Corning Life Sciences, Corning, NY, USA), as previously described (22,23). The difference between the tumor cell migration and invasion ability experiments was that the polycarbonate membrane was either pre-coated or not with Matrigel (30 µl, diluted at 1:3 with cell culture medium; BD Biosciences, San Jose, CA, USA). For the two assays, the cells were starved for 24 h and collected. Cells (5×10⁴) in 200 µl of growth medium containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich) were then placed in the upper chamber. The lower chamber was filled with 600 µl of growth medium containing 20% FBS, and the cells were cultured at 37°C for 48 h. At the end of the experiments, the cells on the upper surface of the filter were removed using a cotton swab, while the cells that migrated or invaded through the filter or Matrigel-coated filter on the lower surface were fixed with 4% neutral-buffered formalin and stained in 0.01% crystal violet solution. The cell numbers were counted in 5 fields (up, down, middle, left and right; magnification x200) for each chamber, and the results were presented as the mean value ± standard deviation (SD). The experiment was performed in triplicate and repeated three times.

The cell adhesion ability was assayed using a 96-well plate that was pre-coated with 30 µl of Matrigel (diluted at 1:3 with growth medium). Particularly, cells were seeded on the plate and incubated in growth medium containing 0.1% BSA overnight. The following day, the cells were washed three times with serum-free medium, resuspended at a concentration of 1×10⁶/ml in serum-free medium, seeded into each well (100 µl/well), and then incubated at 37°C for 1 h. Subsequently, the detached cells were washed away with phosphate-buffered saline (PBS), 10 µl of MTT solution was added to each well containing attached cells, and the plates were incubated at 37°C for 4 h. DMSO (100 µl) was added into each well to dissolve formazan, and the absorbance value was measured at a wavelength of 492 nm using a microplate reader. The data were summarized and presented as the mean value ± SD, and the tumor cell adhesion rate was calculated using the following formula: relative adhesion rate (%) = (A₄₉₂ of experimental group/A₄₉₂ of the control group) × 100%. Each experiment was performed in triplicate and repeated three times.

Athymic nude female BALBC/c mice (4–6 weeks old, 14.96±0.96 g, animal protocol number: 0113061) were obtained from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) [registration no. SCXK(YUE)2008–0002], maintained in specific pathogen-free, temperature-controlled isolation conditions, and fed with sterilized food and autoclaved water. Animal breeding, care, and experimental procedures were approved by the Ethics and Human Committee of Guangzhou Medical University and carried out strictly in accordance with the related regulations on the use of experimental animals.

To evaluate tumor cell peritoneal metastasis ability, 21 athymic nude female BALBC/c mice were randomly assigned to three groups (n=7 per group): i) RhoA-KD xenograft, ii) mock xenograft, and iii) parental xenograft groups. Cells in the logarithmic growth phase were collected and washed twice with PBS. Cells (5.0×10⁶/mouse) in 200 µl of serum-free medium with a survival rate of >95% (assessed using trypan blue staining) were injected into the peritoneal cavity of each mouse. The mice were monitored every 2 days and sacrificed 28 days after tumor cell inoculation. The abdominal circumference and ascetic volume were measured. The number of tumor lesions and disseminated tumors were counted, and the tumor nodules were excised for histopathology, RT-qPCR, western blot analysis, immunohistochemical staining, and terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assays. The liver, spleen, lung, and renal tissues were taken and used for the histopathological analysis of tumor metastasis.

Mouse tumor tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Tissue sections (4 µm) were prepared and stained with hematoxylin and eosin. The consecutive sections were used for immunohistochemical staining. Specifically, the sections were dewaxed in toluene, rehydrated, permeabilized in citrate buffer (pH 6.0), and quenched by 3% H₂O₂ for 15 min to inhibit any endogenous peroxidase activities. The sections were then incubated overnight at 4°C with a polyclonal rabbit anti-RhoA antibody (diluted at 1:100). The following day, the sections were washed with PBS three times and then incubated with a biotinylated goat anti-rabbit IgG (Dako, Glostrup, Denmark), followed by peroxidase-conjugated streptavidin. The colorimetric reaction was performed using diaminobenzidine, and the sections were counterstained with hematoxylin. Immunostained sections were evaluated by two independent observers who were blinded to the tissue identity. The staining was scored as positive vs. Negative when ≥10% tumor cells were positively stained with the polyclonal rabbit anti-RhoA antibody.

Immunohistochemical detection and quantification of apoptosis of mouse xenograft tumor tissues were performed using an In Situ Cell Death Detection kit conjugated with horseradish peroxidase (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. The level of apoptosis was evaluated by counting the TUNEL-positive cells (brown-stained). The apoptotic index was determined as the number of TUNEL-positive cells/total number of cells in five randomly selected high-power fields (magnification, x400).

Data were presented as mean ± SD. Statistical analysis was performed using SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL, USA). Differences among different groups were assessed using one-way analysis of variance, and differences between two groups were assessed using the Student-Newman-Keuls (SNK) test. P<0.05 was considered statistically significant.

In the present study, we assessed the role of RhoA knockdown in ovarian cancer cells. HO8910 cells infected with lentivirus for 48 h showed an infection efficiency of 60% by fluorescence microscopy (Fig. 1A). After the cells were cultured in puromycin (1.5 µg/ml)-containing growth medium for 20 days, stable RhoA-KD and mock ovarian cancer cell populations were successfully generated (Fig. 1A). Although these cell populations were cultured and passaged >10 times, the eGFP-positive expression in the cells remained >98%. The RT-qPCR and western blot analysis confirmed that the RhoA-KD cells had a significantly decreased expression of RhoA mRNA and protein (Fig. 1B and C, P<0.05). Moreover, there was no difference in the RhoA expression between parental HO8910 cells and the mock cells (Fig. 1B and C, P>0.05).

After the stable RhoA-knockdown ovarian cancer cell population was obtained, we first assayed the effects of RhoA silencing on the regulation of tumor cell viability. As shown in Fig. 2A, although there was not a statistical difference in the cell viability among the parental, mock, and RhoA-KD cell cultures on day 1 (P>0.05), the cell viability of the RhoA-KD cultures was significantly attenuated between day 2 and 5 (P<0.05). By contrast, there was no difference in cell viability between the parental HO8910 and mock tumor cells (P>0.05). Moreover, the tumor cell migration and invasion assays showed that the knockdown of RhoA expression reduced the tumor cell migration and invasion abilities compared with the mock and parental HO8910 cells (P<0.05), whereas the parental HO8910 and mock cells showed similar migration and invasion abilities (Fig. 2B and C, P>0.05).

Cancer cell adhesion to the peritoneum is a crucial process and the initial step during ovarian cancer peritoneal metastasis. Therefore, we assessed the effects of the knockdown of RhoA expression on the regulation of tumor cell adhesion. As shown in Fig. 3, the tumor cell adhesion was markedly decreased after RhoA knockdown compared to that of the parental HO8910 and mock cells (P<0.05). However, the mock and parental HO8910 cells showed similar cell adhesion activities (P>0.05).

We assessed the in vivo effect of RhoA silencing in nude mice. We found that 10 days after tumor cell inoculation, the mice with parental or mock cell injections exhibited gradually decreased activity and appeared sluggish, but their abdominal circumference was obviously increased. By contrast, the nude mice with the RhoA-KD cell injection were generally in good condition and their abdominal circumference was gradually increased compared to the remaining two groups (Fig. 4A, P<0.05). Moreover, when the mice were sacrificed at 4 weeks post-inoculation, the tumor formation rate was 71.4% (5/7) in the RhoA-KD group compared to 100% (7/7) in the remaining two groups. The volume of ascites, number of tumor lesions, total number of disseminated tumor nodules, and tumor weight were all signifi-cantly smaller in the RhoA-KD group than in the remaining two groups (Fig. 4B, P<0.05). However, there was no statistically significant difference in tumor growth between the parental and mock groups (P>0.05). In addition, as shown in Fig. 5A, there were more necrotic and apoptotic regions found in tumors with RhoA knockdown than in the parental and mock groups. We performed a TUNEL assay to detect the apoptotic level in these tumor xenografts and found that the apoptotic index was significantly higher in the RhoA-KD group than in the parental and mock groups (Fig. 5E, P<0.05). However, there was no statistically significant difference observed between the parental and mock groups (Fig. 5E, P>0.05).

We confirmed RhoA knockdown in the mouse tumor xenografts using RT-qPCR, western blot analysis and immunos-taining of RhoA mRNA and protein, respectively. The results showed that the RhoA mRNA level was significantly reduced in the RhoA-KD group, compared to the parental and mock groups (Fig. 5B, P<0.05). Similarly, the RhoA protein level was significantly reduced in the RhoA-KD group compared with the parental and mock groups (Fig. 5C and D, P<0.05). However, there was no significant difference of RhoA expression between the parental and mock groups (P>0.05).