The human BEL-7402 hepatocellular carcinoma and HO-8910 ovarian cancer cell lines were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and maintained in RPMI-1640 medium (Gibco-BRL, Rockville, MD, USA) supplemented with 10% fetal calf serum (FCS; Hangzhou Sijiqing Bioengineering Material Co., Ltd., Hangzhou, China), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a 5% CO₂ and 95% air atmosphere and were subcultured at ~80% confluence. Bafilomycin A1 was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell proliferation and viability were analyzed using the WST-1 Cell Proliferation Assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. Briefly, cells were harvested using 0.05% trypsin and suspended in culture medium containing 10% FCS at a concentration of 5×10⁴ cells/ml, and 200 µl suspension was added to each well of a 96-well plate. Cells were cultured for 20 h for adhesion. Bafilomycin A1 was added to the wells at the final concentrations of 200, 400 and 800 nM, in triplicate. At 24, 48 and 72 h, 20 µl WST-1 was added to the cells. Following incubation at 37°C for 4 h, the plates were read to determine the optical density (OD) at 435 nm with 675 nm reference using a spectrophotometer.

Soft agar assays were performed in a 6-well plate. Each well contained 2 ml 0.5% agarose (Sigma-Aldrich) at the bottom and 2 ml 0.35% agarose containing 1×10⁴ cells on top. The medium used was the aforementioned culture media with serial concentrations of bafilomycin A1. Each concentration was set in triplicate. Cells were grown at 37°C in a 5% CO₂ and 95% air atmosphere for 14 days. Colonies were counted under a phase-contrast microscope (XD-202; Nanjing Jiangnan Novel Optics, Co., Ltd., Nanjing, China). The relative proliferation ratio was calculated using the following formula: Relative proliferation ratio = Number of treated/Number of control.

A total of 1×10⁶ BEL-7402 and HO-8910 cells, respectively, were seeded in a 6-well plate (Corning, Inc., Corning, NY, USA) and cultured with the serial concentrations of bafilomycin A1 in RPMI-1640 medium with 10% FCS at 37°C in a 5% CO₂ and 95% air atmosphere for 24, 48 and 72 h. The activities of caspase-3 and −9 in the cellular extracts were determined by colorimetric assay, using commercial Caspase-3 and −9 Activity kits (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The protein concentrations of the samples were determined using a Bicinchoninic Acid Assay kit (Beyotime Institute of Biotechnology). The relative enzyme activity was calculated using the following formula: Relative enzyme activity = enzyme activity of treated cells per µg protein/enzyme activity of control cells per µg protein.

A total of 1×10⁶ BEL-7402 and HO-8910 cells, respectively, were seeded and cultured with the serial concentrations of bafilomycin A1 in RPMI-1640 medium with 10% FCS at 37°C in a 5% CO₂ and 95% air atmosphere for 48 h. The cells were washed with phosphate-buffered saline (PBS), harvested using 0.03% trypsin, re-washed with PBS to remove the trypsin and fixed in 2.5% glutaraldehyde at 4°C overnight. The cells were then centrifuged at 500 × g for 5 min to remove the glutaraldehyde and postfixed in 1% OsO₄ for 2 h. Following postfixation, the cells were washed with PBS, dehydrated in ethanol and embedded in Epon 812 (Sigma-Aldrich). Ultrathin sections were sliced using a Leica-Reichert Ultracut ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany), stained with 2% aqueous uranyl acetate (Sigma-Aldrich) and lead citrate (Sigma-Aldrich) and observed with an HT-7700 electron microscope (Hitachi, Ltd., Tokyo, Japan).

The cells were cultured in the aforementioned complete culture medium until reaching 80% confluence and then starved in serum-free medium for 8 h. The cells were subsequently washed with PBS, harvested, diluted to the concentration of 1×10⁵ cells/ml in 0.1% serum RPMI-1640 medium with serial concentrations of bafilomycin A1 and then plated in an invasion chamber (1×10⁴ cells in 100 µl medium per well) of a Cell Invasion Assay kit (Chemicon International, Inc., Temecula, CA, USA). The invasion chamber was inserted into the feeder tray, which contained 150 µl 10% serum RPMI-1640 medium per well. The plate was incubated for 20 h at 37°C in 5% CO₂ and 95% air. The cells that had invaded through the membrane were washed with detachment solution and stained with lysis buffer/dye solution, and the fluorescence absorbance was measured in a luminescence spectrometer at 480/520 nm.

Total RNA of the control cells and cells that had been exposed to 400 nM bafilomycin A1 for 48 h was extracted using TRIzol® reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. RNA concentration and purity were determined via ultraviolet spectrometry, measuring the OD at 260 and 280 nm.

Total extracted RNA was subjected to mRNA and miRNA microarray using the Affymetrix GeneChip® Human Gene and miRNA Arrays (Affymetrix, Inc., Santa Clara, CA, USA), respectively, at Genminix Informatics Ltd., Co. (Shanghai, China) according to the instructions provided with the Affymetrix arrays. Briefly, RNA was extracted from cells following treatment with 400 nM bafilomycin A1 for 48 h using TRIzol reagent (Invitrogen, Life Technologies). The quantity and purity of the RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). The purity and integrity of each extracted RNA met the following requirements: A260/A280 >1.6, A260/A230 >1 and RNA Integrity Number >5. The small RNA fraction indicated an abundance of RNA <200 nt, compared with total RNA using Agilent RNA 6000 Nano assay (Agilent Technologies, Inc., Santa Clara, CA, USA), and was acceptable for miRNA assay. The possibility of genomic DNA contamination was excluded using gel electrophoresis. A total of 2 µg RNA from each group was respectively converted into cyanine-5-labeled target cRNA, hybridized to the Affymetrix GeneChip Human Gene and miRNA Arrays, respectively, using the Affymetrix GeneChip Fluidics Station 450 (Affymetrix, Inc.), and scanned with an Affymetrix GeneChip Scanner 3000 7G (Affymetrix, Inc.). Following normalization, differentially expressed mRNAs or miRNAs were established at log2 (fold change) >1 and P<0.05.

RNA was transcribed into cDNA using the miScript II RT kit (Qiagen, Hilden, Germany). The reaction components were as follows: Total RNA, 1 µg; miScript HiSpec Buffer, 4 µl; Nucleic Acid Mix, 2 µl; miScript Reverse Transcriptase Mix, 2 µl; RNase-free H₂O, <20 µl. The reaction was performed at 37°C for 60 min and 95°C for 5 min on an ABI PCR 9700 system (Applied Biosystems, Foster City, CA, USA). cDNA was diluted in 80 µl nuclease-free H₂O for the addition of LightCycler® 480 SYBR Green I Master (Roche Diagnostics, Indianapolis, IN, USA). The reaction system for the qPCR was as follows: LightCycler 480 SYBR Green I Master, 5 µl; forward primer, 0.2 µl; reverse primer, 0.2 µl (provided by RiboBio Co., Ltd., Guangzhou, China); cDNA, 1 µl; nuclease-free H₂O, 3.6 µl. The PCR was run using an ABI 7500 Fast system (Applied Biosystems), as follows: 94°C for 2 min, followed by 40 cycles of 94°C for 15 sec, 62°C for 40 sec and 70°C for 40 sec. The mRNA expression was normalized against the expression of U6. All samples were analyzed in triplicate. Relative expression was calculated using the comparative threshold cycle (Ct) method and was indicated as the fold change, as follows: Fold change = 2^{−(ΔCttreated - ΔCtcontrol)}.

Microarray data were analyzed by Genminix Informatics Ltd., Co. The integrated analysis of the differential mRNA and miRNA expression due to treatment with bafilomycin A1 was performed to reveal the target genes of the drug via miRNA regulation. The target genes of the differential miRNA were predicted using bioinformatics [TargetScan (http://www.targetscan.org/) and MicroCosm Targets (http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets)]. The overlapping altered genes in the mRNA microarray and the miRNA predicted genes were subjected to Gene Ontology (GO) term and pathway analyses, in order to organize the genes into hierarchical categories and uncover the altered pathways and miRNA-gene regulatory network upon which bafilomycin A1 likely has an effect.

Statistical analysis was carried out using Microsoft Excel software (Microsoft Corp., Redmond, WA, USA). Data were compared using a two-tailed Student's t-test, and P<0.05 was considered to indicate a statistically significant difference.

WST-1 cell proliferation and soft-agar colony formation assays were performed to determine the effect of bafilomycin A1 on BEL-7402 and HO-8910 cells. The results indicated that the proliferation of the two cell lines was inhibited by bafilomycin A1, as shown in Fig. 1A. The inhibitory effect was also evident in the soft-agar colony formation assay (Fig. 1B). Compared with the control cells, few micro- or middle-colonies (containing 3–5 or 6–10 cells/colony, respectively) and non-macro-colonies (containing >10 cells) could be observed among the bafilomycin A1-treated cells, and the total numbers of colonies were significantly reduced.

TEM was used to capture images of the cells treated with various concentrations of bafilomycin A1 at 48 h. Non-bafilomycin A1-treated BEL-7402 and HO-8910 cells are shown in Fig. 2A and B, respectively. BEL-7402 and HO-8910 cells treated with 100 nM bafilomycin A1 (Fig. 2C and D, respectively) exhibited indentations and folding of the nuclear membrane, whereas BEL-7402 and HO-8910 cells treated with 200 nM bafilomycin A1 (Fig. 2E and F, respectively) exhibited chromosome condensation at the periphery of the nuclear membrane, condensed nuclei and vacuolar cytoplasms. The images suggested an apoptotic response to the cellular toxicity. Furthermore, the assays for capsase-3 and −9, which are two important enzymes involved in apoptosis, showed significant increases following bafilomycin A1-treatment (Fig. 2G).

According to the cell invasion assays, the invasive potential of the BEL-7402 and HO-8910 cells was significantly suppressed with the bafilomycin A treatment, as shown in Fig. 3.

The numbers of altered mRNAs and miRNAs in BEL-7402 and HO-8910 cells 48 h after exposure to 400 nM bafilomycin A1 are shown in Table I. The overlapping altered genes in the mRNA microarray and the miRNA predicted genes were determined, and GO analysis was performed. The altered pathways are shown in Fig. 4A. After treatment with 400 nM bafilomycin A1 for 48 h, the following miRNAs were altered in the two cell lines: miR-923, miR-1246, miR-149*, miR-638 and miR-210 were significantly upregulated, whereas miR-99a, miR-181a-2* and miR-339-5p were significantly downregulated. These results were confirmed by qPCR (Fig. 4B).