All samples were collected from the inpatient clinic of Shanghai Pudong New Area Gongli Hospital (Shanghai, China) between June 2013 and December 2014. All human materials were obtained according to consent regulation and approved by the Ethics Review Committee of the World Health Organization of Collaborating Center of Research in Human Production authorized by Shanghai Municipal Government. Briefly, 4 µl of CD44⁺ and CD117⁺ primary monoclonal antibodies (mouse anti-human CD117-FITC, rabbit anti-human CD44-PE; eBioscience) at 1 mg/100 ml were added to the primary ovarian cancer cell sediment to block nonspecific binding, and the cells were stored at 4°C in phosphate-buffered saline (PBS) for 30 min in a volume of 1 ml as previously described (1,3,4,9). After the reaction, the cells were washed twice in PBS. They were isolated and enriched by flow cytometry (BD FACSAria; BD Biosciences, San Jose, CA, USA), incubated at 10°C in PBS for 15 min before being washed twice in PBS. Single cells were plated at 1,000 cells/ml in Dulbecco's modified Eagle's medium (DMEM):F12 (HyClone), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml epidermal growth factor (EGF), 5 µg/ml insulin and 0.5% bovine serum albumin (BSA) (all from Sigma-Aldrich). All CD44⁺/CD117⁺ cells were cultured in the above conditions as non-adherent spherical clusters, termed ovarian carcinoma-initiating cells. All cells were cultured under the same conditions until passage three, when they were used for further experiments.

Total RNA from each cell was isolated using TRIzol reagent (Invitrogen), according to the manufacturer's protocol. The RNA samples were treated with DNase I (Sigma-Aldrich, St. Louis, MO, USA), quantified and reverse-transcribed into cDNA using the ReverTra Ace-α First Strand cDNA Synthesis kit (Toyobo). Quantitative RT-PCR was conducted using a RealPlex4 real-time PCR detection system from Eppendorf Co., Ltd. (Germany), with SYBR-Green Real-Time PCR Master Mix (Toyobo) as the detection dye. Amplification was performed over 40 cycles with denaturation at 95°C for 15 sec and annealing at 58°C for 45 sec. Target cDNA was quantified using the relative quantification method. A comparative threshold cycle (Ct) was used to determine gene expression relative to a control (calibrator) and steady-state mRNA levels are reported as an n-fold difference relative to the calibrator. For each sample, the maker gene Ct value was normalized using the formula Ct = Ct_genes-Ct_18sRNA. To determine relative expression levels, the following formula was used: ΔΔCt = ΔCt_all_ groups-ΔCt_blank control_group. The values used to plot relative expression of markers was calculated using the expression 2^−ΔΔCt. The mRNA levels were calibrated based on levels of 18s rRNA. The cDNA of each gene was amplified using primers as previously described (12,13).

Northern blotting experiments were performed as previously described (7,13). For all samples, 20 µg of good quality total RNA was analyzed on a 7.5 M urea 12% polyacrylamide denaturing gel and transferred to a Hybond N⁺ nylon membrane (Amersham, Freiburg, Germany). Membranes were cross-linked using UV light for 30 sec at 1,200 mJ/cm². Hybridization was performed with the anti-sense starfire probe to be detected the lncRNA BACE1-AS fragments according to the manufacturer's instructions (12,13). After washing, membranes were exposed for 20–40 h to Kodak XAR-5 film (Sigma-Aldrich). As a positive control, all membranes were hybridized with a human U6 snRNA probe, 5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3′. Exposure times for the U6 control probe varied between 15 and 30 min.

Cells were seeded (2×10³/well) in 96-well plates and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂, until 85% confluent. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Sigma Chemicals reagent (5 mg/ml) was added to the maintenance cell medium at different time points, and incubated at 37°C for an additional 4 h. The reaction was terminated with the addition of 150 µl dimethylsulfoxide (DMSO; Sigma Chemicals) per well. Cells were lysed for 15 min, and the plates were gently shaken for 5 min. Absorbance values were determined using the enzyme-linked immunosorbent assay (ELISA) reader (Model 680; Bio-Rad) at 490 nm.

Total protein extracts were resolved by 12% SDS-PAGE and transferred on polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking, the PVDF membranes were washed four times for 15 min with Tris-buffered saline with Tween-20 (TBST) at room temperature and incubated with primary antibody (Table I). Following extensive washing, membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG (1:1,000; Santa Cruz Biotechnology) for 1 h. After washing four times for 15 min with TBST at room temperature, the immunoreactivity was visualized by enhanced chemiluminescence (ECL kit; Pierce Biotechnology), and membranes were exposed to Kodak XAR-5 film.

All steps were performed as previously described in 6-well plates (7). The base layer of each well consisted of 2 ml with final concentrations of 1X media (DMEM+10% FBS) and 0.6% low melting point agarose. Plates were chilled at 4°C until solid and then a 1 ml growth layer of agar was poured, consisting of 1×10⁴ cells suspended in 1X media and 0.3% low melting point agarose. Plates were again chilled at 4°C until the growth layer congealed. An additional 1 ml of 1X media without agarose was added on top of the growth layer on day 0 and again on day 15 of growth. Cells were allowed to grow at 37°C for 1 month and then the total number of colonies was counted. Assays were repeated three times. Results were statistically analyzed by paired t-test using the GraphPad Prism program.

All steps were performed as previously described (1). Briefly, cells (2×10⁵) were resuspended in 200 µl of serum-free medium and seeded on the top chamber of the 8.0-µm pore, 6.5 mm polycarbonate Transwell filters (Corning). A total of 600 µl of DMEM+10% FBS was added to the bottom chamber. The cells were allowed to migrate for 24 h at 37°C in a humidified incubator with 5% CO₂. The cells attached to the lower surface of the membrane were fixed in 4% paraformaldehyde at room temperature for 30 min and stained with 4,6-diamidino-2-phenylindole (DAPI) (C1002; Beyotime Institute of Biotechnology, China). The number of cells on the lower surface of the filters was counted under the microscope. A total of five fields were counted for each Transwell filter.

Apoptosis was evaluated using an Annexin V-FITC/PI staining kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions and as previously described (16). Briefly, cells were seeded (3×10⁵/well) in 6-well plates and cultured until 85% confluent. Cells were washed with PBS three times, and collected by centrifugation (Allegra X-22R; Beckman Coulter) at 1,000 × g for 5 min. The cell pellets were resuspended in 0.1 ml of PBS, stained with the Annexin V-FITC/PI kit, incubated for 30 min at 4°C and analyzed using a fluorescence-activated cell sorting (FACS) machine (BD FACSAria). A total of 20,000 events were acquired for analysis using CellQuest software.

The cultured cells were washed three times with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. After blocking, the cells were incubated first with primary antibody (Table I) overnight at 4°C, and then with FITC-conjugated goat anti-rabbit IgG antibody (1:200; Abcam, Cambridge, UK) and 5 µg/ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Cells were thoroughly washed with TBST and viewed through a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA).

OCSCs were divided into two groups: a negative control group (OCSCs pretreated with DMSO), which were treated with DMSO for 72 h; and an experimental group (OCSCs pretreated with anisomycin), which were treated with anisomycin (at its half maximal inhibitory concentration, IC₅₀=8.5 µmol) for 72 h. Approximately 1×10⁵ logarithmically growing OCSCs were inoculated in BALB/c nude, or nude mice. Each experimental group consisted of seven mice. After seven weeks of observation, the mice were sacrificed and tumors were stripped (7,8). Tumor weight was measured and tumor volume was calculated according to the formula: Tumor volume (mm³) = (ab²)/2, where a, represents the longest axis (mm) and b, the shortest axis (mm).

Each experiment was performed as least three times and data are shown as the mean ± SE where applicable. Differences were evaluated using Mann-Whitney test. The probability p<0.05 was considered to indicate a statistically significant result.

We examined a total of seven ovarian cancer patients with undifferentiated and mixed epithelial ovarian carcinoma. The characteristics of all ovarian patients are summarized in Table II. The age, stage, grade of differentiation and histology distribution of the patient populations were released by the International Federation of Gynaecology and Obstetrics (FIGO) annual report for European countries (1,7).

Cell viability (MTT) assays revealed no significant difference in the viability of anisomycin (8.5 µmol) treated OCSCs compared to controls in the first 24 h of treatment (i.e., at 0, 12 and 24 h; Fig. 1A). However, over time (48–72 h), anisomycin-treated OCSCs showed a significant decrease in cell proliferation compared to the DMSO treated and untreated group (Fig. 1A). After 72 h of treatment, there was a significant increase in the percentage of cells undergoing early apoptosis (Annexin V-FITC⁺/PI⁻) in the anisomycin-treated OCSCs compared to the untreated OCSCs (Fig. 1B). Moreover, there were significantly fewer invading aniso-mycin-treated OCSCs/well (migration cell number, 63±13) than DMSO-treated OCSCs/well (102±16) or untreated OCSCs/well (115±15) (Fig. 1C). After 72 h of treatment, there were substantially fewer colonies in the anisomycin-treated OCSC agar plate (33.01±9.17%) when plated at low density compared to the DMSO-treated OCSC plate (76.71±6.26%) or the untreated OCSC plate (82.26±8.32%) (Fig. 1D). These results suggest that anisomycin suppresses OCSC proliferation and invasion.

Our qRT-PCR analysis showed that the expression of lncRNA BACE1-AS in the anisomycin-treated OCSCs was significantly elevated compared to the DMSO treated OCSCs or untreated OCSCs after 72 h (Fig. 2A). In addition, the expression of BACE1, APP, Bcl2 and Bax mRNAs in the anisomycin treated OCSCs was elevated compared to the DMSO treated OCSCs or untreated OCSCs after 72 h (Fig. 2B). Expression of the Ki67 (a cellular marker for proliferation) mRNA in the anisomycin treated OCSCs was decreased compared the DMSO treated or untreated OCSCs (Fig. 2B).

In terms of protein expression, our western blot analysis confirmed that expression of amyloid-β-related proteins (BACE1 and Aβ1-42) were significantly increased in the anisomycin treated OCSCs compared to the DMSO treated or untreated OCSCs after 72 h (Fig. 3A and B). However, the expression of the Ki67 protein was significantly decreased in the anisomycin treated OCSCs compared the DMSO treated or untreated OCSCs (Fig. 3A and B). Additionally, IF staining confirmed that BACE1 and Aβ1-42 levels increased in the anisomycin treated OCSCs compared the DMSO treated or untreated OCSCs after 72 h (Fig. 3C). These data indicate that anisomycin induces lncRNA BACE1-AS expression and expression of amyloid-β-related proteins.

All mice inoculated with anisomycin or DMSO pretreated OCSCs developed tumors ~7 weeks after injection. Although both experimental groups eventually developed tumors, tumors formed by anisomycin pretreated OCSCs grew more slowly than those derived from DMSO pretreated OCSCs (Fig. 4A). Furthermore, when mice were sacrificed seven weeks after inoculation with OCSCs, tumors obtained from the anisomycin pretreated group were significantly smaller and lighter than those from the DMSO pretreated group (Fig. 4B and C). Representative hematoxylin and eosin-stained sections of all subcutaneous xenograft tumors were categorized as mixed epithelial ovarian carcinoma of moderate or poor differentiation (grade II/III) (Fig. 4D). Furthermore, immu-nohistochemical staining indicated that expression of the BACE1 and Aβ1-42 proteins were significantly higher in tumors derived from anisomycin pretreated OCSCs compared to those derived from the DMSO pretreated OCSCs (Fig. 4D). The expression of the Ki67 protein was significantly decreased in the anisomycin pretreated OCSCs compared to the DMSO pretreated OCSCs (Fig. 4D). Thus, xenografts formed by anisomycin pretreated OCSCs were smaller and experienced reduced proliferation compared to those formed by DMSO pretreated OCSCs.

To evaluate whether anisomycin regulates proliferation and invasion of OCSCs by inhibiting lncRNA BACE1-AS expression, we used a siRNA targeting BACE1-AS. OCSCs were transfected with either siRNA-BACE1-AS or siRNA-Mock and then treated with anisomycin for 72 h. The MTT assay indicated that the survival ratio of the anisomycin treated siRNA-BACE1-AS transfected OCSCs group was significantly higher than the anisomycin treated siRNA-Mock transfected OCSCs group or the anisomycin treated control group (Fig. 5A). Moreover, western blot analysis showed that BACE1 protein expression was significantly lower in the anisomycin treated siRNA-BACE1-AS transfected OCSC group compared to the anisomycin treated siRNA-Mock transfected OCSC group or the anisomycin treated control group (Fig. 5B). Finally, northern blot analysis revealed that the hybridization signal of lncRNA BACE1-AS from the anisomycin treated OCSCs after 72 h was signifi-cantly stronger than from the DMSO treated or untreated OCSCs (Fig. 5C); but the hybridization signal of lncRNA BACE1-AS from the anisomycin treated siRNA-BACE1-AS transfected OCSC group was significantly lower than in the anisomycin treated siRNA-Mock transfected OCSC group or the anisomycin treated control group (Fig. 5D). These results indicate that silencing of lncRNA BACE1-AS expression can weaken the effects of anisomycin in OCSCs.