The OC cell lines A2780, A2780-PM and A2780-PTX were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. The cells were harvested by centrifugation, rinsed with phosphate-buffered saline (PBS), and were then used for extraction of total RNA.

The resistant ovarian cancer cell line A2780-PTX was cultured in the top chambers of Matrigel™-coated Transwell^® inserts (Beckon-Dickinson Biosciences) containing 1 ml of DMEM containing 10% FBS in the lower chamber. A2780-PTX cells that were highly invasive were able to pass through the membrane to the lower chamber. These cells have high resistance and high invasiveness, and were termed A2780-PTX-PM. Flow cytometry was used to determine the OCSCs from this population. Briefly, the cells were trypsinized to prepare a single cell suspension, adjusting the cell concentration to 5×10⁵/tube cells. Then, 2 ml of diluted CD117 antibody was added to each tube containing the cell suspension, the mixture was incubated at room temperature for 30 min, washed thrice with PBS, and centrifuged at 1,000 rpm for 5 min. The cells were then incubated with fluorescein isothiocyanate (FITC)-labeled secondary antibodies at room temperature for 30 min, washed thrice with PBS, centrifuged at 1,000 rpm for 5 min, and resuspended in 2 ml PBS, prior to detection of fluorescence intensity and cell sorting. The sorted cells were cultured after the completion of the relevant experiments.

Cell proliferation was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were directly seeded into 96-well plates (5×10³ cells/well) and allowed to adhere. At different time points (0, 24, 48 and 72 h) after seeding, 20 µl of 5 mg/ml of MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells, and the cells were incubated at 37°C for 4 h. The supernatants were removed, and 150 µl of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well. The absorbance of each well was measured at 490 nm.

Cells were seeded at 1×10⁶ cells/well in 6-well culture plates and cultured to confluence. The cell monolayer was then scratched with a pipette tip (200 µl). Cells were washed thrice with PBS and cultured in an FBS-free medium. The cells were photographed at 0, 24 and 48 h, and the scratched areas were measured using ImageJ software. The wound healing rate was calculated using the following formula: Wound healing rate = (area of original wound - area of actual wound at different times)/area of original wound × 100%.

First, 5×10⁴ cells were resuspended in FBS-free DMEM and seeded into the top chambers of Matrigel™-coated Transwell^® inserts. The lower compartment of the chamber contained 10% FBS as a chemoattractant. After incubation for 48 h at 37°C in a 5% CO₂ atmosphere, the cells on the upper surface of the membrane were wiped away. Cells on the lower surface of the membrane were washed with PBS, fixed in 4% methanol and stained with crystal violet dye to quantify the extent of invasion.

Total RNA was extracted from OC cell lines using TRIzol reagent (Takara Bio, Japan). Real-time RT-PCR was carried out with 2 µg of total RNA using AMV reverse transcriptase and random primers (Takara Bio). PCR primers were designed according to the sequences in GenBank. cDNA amplification was carried out with a SYBR™ Premix Ex Taq II kit (Takara), using glyceraldehyde 3-phosphate dehydrogenase (18s) as an internal control, according to the manufacturer's instructions. Briefly, cDNA amplification for each primer was carried out in a final volume of 20 µl containing 10 µl SYBR Premix Ex Taq (2X), 0.08 µl of each primer, 0.4 µl of ROX reference dye, and 1 µl of template cDNA (50 µg/µl). The PCR protocol was as follows: initial incubation at 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 34 sec. The relative gene expression levels (amount of the target gene normalized to that of the endogenous control gene) were calculated using the comparative Ct method (2^−ΔΔCt).

For the immunofluorescence experiment, the cells were cultured on glass coverslips, fixed with PBS containing 4% formaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 30 min at room temperature. After washing with PBS, the cells were incubated overnight at 4°C with antibodies to RhoC, CD117 and CD133 (1:50; Proteintech, Shanghai, China). Then, the cells were washed three times with PBS and treated with the secondary antibody for 2 h at room temperature in a dark and humidified chamber. After washing thrice with PBS, the immunostained cells were mounted in mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) for 5 min, and washed thrice with PBS. The cells were then visualized under a fluorescence microscope equipped with a camera.

Ranked data were analyzed using Spearman's rank correlation coefficient. The Mann-Whitney U test was used to differentiate between the mean values of different groups. A P-value of <0.05 was considered to indicate a statistically significant result. SPSS v.17.0 (IBM, Armonk, NY, USA) was employed to analyze data.

A2780-PM and A2780-PTX-PM cells showed higher cell proliferation (Fig. 1A), drug resistance (Fig. 1B and C) and invasion and migration ability (Fig. 2) than these parameters in the A2780 and A2780-PTX cell lines. Additionally, CD133 and CD117 expression levels were higher in the A2780-PM and A2780-PTX-PM cells than these levels in the A2780 and A2780-PTX cells (Fig. 3A and B).

Cells were sorted through flow cytometric analysis to select ovarian cancer cells with stem cell-like characteristics (Fig. 4).

OCSCs showed induced cell proliferation, drug resistance and migration, and invasion ability. si-RhoC transfection in OCSCs reduced cell proliferation (Fig. 5A), drug resistance (Fig. 5B and C), and migration and invasion ability (Fig. 6). Results of the immunofluorescence analysis revealed that the expression of RhoC, CD133 and CD117 were also induced in the OCSCs, while transfection with si-RhoC reduced the expression of RhoC, CD133 and CD117 (Fig. 3C-E).

We assessed the mRNA expression levels of RhoC, CD133, CD117, MDR1 and MMP9 in A2780-PTX-PM cells, OCSCs and si-RhoC-transfected OCSCs. The results revealed that the expression levels of all of these markers were higher in OCSCs than levels in the A2780-PTX-PM cells, but the expression levels were lower in the si-RhoC-transfected OCSCs than levels in the OCSCs (Fig. 7).