Human ovary cancer cell line SKOV3 was a gift from the Cancer Research Institute of Southern Medical University; cisplatin resistant human ovary cancer cell line (SKOV/DDP) was a gift from the Central Laboratory, Qingdao University Medical College Affiliated Hospital. Female BALB/c-nu/nu nude mice (aged 4–5 weeks) were purchased from the Experimental Animal Center, Zhongshan University, under permit number SCKK (Guangdong) 2011–0029 and housed in the Animal Center on the North Campus of Zhongshan University. The animal experiments were approved by the Animal Care and Use Committee of Southern Medical University.

SKOV3 and SKOV3/DDP cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) at 37°C and 5% CO₂ in incubator with saturated humidity.

SKOV3/DDP cells were transfected with lentivirus containing miR-100 mimics, (LV-miR-100), antisense (LV-anti-100) and empty vector (LV-NC) (Shanghai GenePharma, Co., Ltd., Shanghai, China). After 12 h, virus solution was removed and the cells were cultured in RPMI-1640 containing 10% FBS for another 48 h and examined for infection efficiency using a fluorescence microscope, and further confirmed using real-time quantitative PCR (qRT-PCR). The cells were cultured in complete culture medium containing puromycin (2 µg/ml) for two weeks to obtain SKOV3/DDP cells with miR-100 stably downregulated or upregulated.

RNA (3 µg) was reverse transcribed using random primers (Applied Biosystems, Carlsbad, CA, USA). miRNA expression was determined using TaqMan^® miRNA assays (Applied Biosystems) according to the manufacturers protocol. In brief, 50 ng of total RNA was reverse transcribed using specific miRNA primers. U6 expression was used for normalization using the comparative CT method (14). The primers (Shanghai GenePharma) were forward, 5-ATCATTAAAC CCGTAGATCCGAA and reverse, 5-GGAACGCTTCACGA ATTTG for miR-100; and forward, 5-ATTGGAACGATACA GAGAAGATT and reverse, 5-GGAACGCTTCACGAATT TG for U6.

Transfected SKOV3/DDP cells were seeded in wells of 96-well plates at a density of 3×10³ cells/well, cultured overnight and added with different concentrations of cisplatin from 0 (control) to 64 µg/ml. For each concentration, 5-wells were used. The cells were cultured for 48 h, added with 10 µl CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) and cultured for another 4 h to measure the absorbance (A) at 450 nm. The values were used to calculate the IC₅₀ of cisplatin. In addition, the transfected SKOV3/DDP cells were seeded in wells of 96-well plates at a density of 2×10³ and measured for absorbance at 450 nm wavelength at different time-points (12–72 h) using a plate reader to plot growth curves.

Total protein was extracted 72 h after transfection, separated on SDS-PAGE, transferred to membrane and incubated with primary antibodies (rabbit anti-human PLK1, anti-human mTOR and anti-human GAPDH monoclonal antibodies; Cell Signaling Technology, Beverly, MA, USA) for 1 h, and secondary antibody (goat anti-rabbit IgG; Abcam, Cambridge, UK) for 1 h. The membrane was developed using enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA) and the gray value of the bands was quantified using Quantity One software to calculate the relative expression of mTOR and PLK1. The experiments were repeated 3 times.

SKOV3/DDP cells with stably upregulated and downregulated miR-100 expression and control cells were cultured for 48 h, harvested and stained with FITC Annexin V and propidium iodide (PI) (FITC Annexin V apoptosis detection kit; BD Biosciences, San Jose, CA, USA) for cell cycle analysis, they were fixed in 70% ethanol on ice, washed with phosphate-buffered saline (PBS), then stained with PI containing RNase A (20 µg/ml in PBS). The cells were analyzed on FACSVerse flow cytometry (BD Biosciences).

Cells were inoculated in wells of 6-well plates at a density of 1×10² cells/well and cultured for 14 days in RPMI-1640 medium with 10% FBS, fixed in methanol fixed, stained with 0.1% crystal violet solution. Samples were photographed and counted for the number of visible colonies.

Cells in logarithmic growth were adjusted to a cell density of 5×10⁷/ml and injected subcutaneously into the nude mice at 200 µl/mouse. When the tumors grew to 5 mm in diameter cisplatin was injected at 4 mg/kg every 3 days for a total of 7 times. The size of tumor was measured every 3 days to draw the growth curve. Three days after the end of treatment, the mice were sacrificed, tumor tissue removed, fixed in 10% formalin and used in immunohistochemistry assays for PLK1 and mTOR.

The SPSS 20 software was used for statistical analysis, measurement data were expressed as mean ± standard derivation (SD). Paired groups were analyzed using Students t-test, and one-way analysis of variance (ANOVA) was applied to compare the difference among multiple groups. Values with P<0.05 was considered statistically significant.

We first determined the IC₅₀ to measure the resistance to cisplatin. For SKOV3/DDP and SKOV3 cells, IC₅₀ was 8.29 and 3.72 µg/ml, respectively. Therefore, SKOV3/DDP was 2.23 times more resistant to cisplatin than SKOV3 (Fig. 1A).

We then profiled the expression of miR-100 in the two cell lines. It was found that the expression of miR-100 was 25 times less in SKOV3/DDP cells as compared with SKOV3 cells (Fig. 1B). However, after transfection with the miR-100 mimics, the level of miR-100 in transfected SKOV3/DDP cells was significantly higher than that in LV-NC transfected cells (P<0.05; Fig. 2A), while transfection with the miR-100 antisense resulted in significantly reduced miR-100 level in SKOV3/DDP cells (P<0.01; Fig. 2A).

The sensitivity of resistant SKOV3/DDP cells to cisplatin was determined after the cells were upregulated by miR-100 mimics. Compared with the cells transfected with LV-NC, cells transfected with LV-miR-100 had significantly low IC₅₀ to cisplatin (P<0.001). On the other hand, when transfected with LV-anti-100, the IC₅₀ was significantly increased (P<0.001; Fig. 2B).

Next, we examined the expression of mTOR and PLK1 in the cell lines before and after transfection using western blot analysis. Compared with SKOV3 cells, mTOR and PLK1 protein levels were significantly higher in the resistant SKOV3/DDP cells (P<0.05; Fig. 3A). After transfection with LV-miR-100, the levels of the two proteins were significantly lower than these in the LV-NC-transfected cells (P<0.05; Fig. 3B), while transfection with LV-anti-100 resulted in significantly higher expression of the two proteins (P<0.05; Fig. 3B).

The numbers of colony were reduced or increased after SKOV3/DDP cells were transfected with LV-miR-100 or LV-anti-100, respectively (Fig. 4A and B). After the transfection with LV-miR-100, the growth of SKOV3/DDP cells was reduced as revealed by the proliferation index (Fig. 4C); on the other hand, transfection with LV-anti-100 increased the proliferation index, as compared with LV-NC (Fig. 4C).

After 48 h of cisplatin treatment at 3 µg/ml, we examined the early apoptosis rate in SKOV3/DDP cells. The results showed that the apoptosis rate was significantly higher after the cells were transfected with LV-miR-100 as compared with LV-NC-transfected cells (P<0.01), while the apoptosis rate in SKOV3/DDP cells transfected with LV-anti-100 was significantly lower (P<0.05; Fig. 5A and C).

The proportions of G1 and S phase cells in LV-miR-100- transfected cells was significantly higher or lower than those in LV-NC-transfected cells (P<0.01; Fig. 5B and D), respectively; in contrast, the proportions of G1 and S phase cells in LV-anti-100-transfected cells were significantly lower or higher than those in LV-NC-transfected cells (P<0.05; Fig. 5B and D), respectively.

We then used mouse models with grafted cells to assess the efficacy of cisplatin. When the mice were grafted with SKOV3/DDP cells transfected with LV-miR-100, the tumors grew slower than those grafted with LV-NC-transfected SKOV3/DDP cells. On other hand, tumors grew faster in mice grafted with LV-anti-100 than with LV-NC-transfected SKOV3/DDP cells (Fig. 6A). Twenty-one days after cisplatin treatment, the tumor volumes were smaller from SKOV3/DDP cells stably transfected with LV-miR-100 than with LV-NC (Fig. 6A and B; P<0.01). In contrast, the tumors derived from SKOV3/DDP cells stably transfected with LV-anti-100 were significantly larger than with LV-NC (P<0.01; Fig. 6A and B). Immunohistochemical analysis indicated that compared with tumors derived from LV-NC transfected cells, mTOR and PLK1 were downregulated in the tumors derived from LV-miR-100 transfected-cells and upregulated in the tumors derived from LV-anti-100 transfected cells (P<0.05; Fig. 6C), respectively.