The human breast cancer cell lines (MCF-7 and MDA-MB-231), and human breast cells (HCC1937) purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China) were used. Additional instruments and reagents used are shown in Table I. MCF-7, MDA-MB-231 and HCC1937 cells were removed from storage in liquid nitrogen and thawed in a water bath set to 37°C. Cells were then added to culture medium [Dulbecco's modified Eagle's medium/F12 (DMEM/F12) supplemented with 5% fetal bovine serum (FBS), 0.1 mg/ml streptomycin, 100 U/ml penicillin and 2 mmol/l glutamine]. Cells were grown in culture bottles in an incubator (37°C, 5% CO₂) for 48 h. Culture medium was removed when cells reached 90% confluence. Cells were trypsinized in 0.25% trypsin, and centrifuged at 800 × g for 10 min at room temperature. Cells were washed with DMEM/F12 and then seeded again in cell culture bottles.

TRIzol was added to cell lysis buffer for lysis of MCF-7, MDA-MB-231 and HCC1937 cells that were in the logarithmic growth phase. After 5 min of digestion, lysates were placed in new Eppendorf (EP) tubes (Corning, Inc., Corning, NY, USA), and 200 µl chloroform was added. Solutions were shaken up and down 15 times, and placed at room temperature for 5 min. Samples were then centrifuged (8,500 × g, 4°C, 10 min). RNA in the supernatant was transferred to new EP tubes. Next, 75% ethanol was added and samples were centrifuged (6,500 × g, 4°C, 5 min). The supernatant was discarded, and solutions were placed on a super-clean worktable to air dry. DEPC water (Biosharp, Hefei, China) was then added and mixed well. The concentration and purity of RNA were determined by UV spectrophotometry. According to the instructions of the reverse transcription kit, RNA was reverse transcribed into cDNA. Real-time PCR amplification of cDNA was performed to measure the expression of FTO mRNA in each group of cells.

After trypsinization of MDA-MB-231 cells in the logarithmic growth phase, cells were washed with DMEM/F12, cell growth medium was added, and the cell concentration was adjusted to 2×10⁵/ml. Cells were then seeded in 6-well culture plates, and placed at 37°C, 5% CO₂ for 48 h. When cells were 50% confluent, growth medium was replaced with incomplete culture medium (without FBS), and placed at 37°C, 5% CO₂ for 1 h. The incomplete, serum-free medium was mixed with miFTO inhibitor or inhibitor control, and incubated at 37°C for 5 min (solution A). The serum-free medium was mixed with Lipofectamine 2000 (solution B) (KeyGen, Nanjing, China). Next, solutions A and B were mixed, and left to incubate at room temperature for 20 min. The cell culture medium was discarded, and cells were repeatedly washed with phosphate-buffered saline (PBS). The transfection reagent and miRNA were added to the cell culture plates, and cells were placed at 37°C, 5% CO₂ for 6 h. The medium was replaced with complete culture medium, and cells were left in the incubator for an additional 48 h.

MDA-MB-231 cells were transfected with miFTO inhibitor, or inhibitor control and left to incubate at 37°C, 5% CO₂ for 48 h. Culture supernatant was harvested and transferred to EP tubes, and centrifuged (800 × g, 5 min). The content of lactic acid in supernatant was measured according to the instructions of the Sigma Lactic Acid Test kit (Sigma-Aldrich, St. Louis, MO, USA).

Transfected MDA-MB-231 cells grown in the incubator (37°C, 5% CO₂) for 48 h were trypsinized and washed with PBS. Cell culture medium was added to resuspend the cells, then cells were transferred to EP tubes and centrifuged (800 × g, 5 min). The supernatant was discarded. Cells were washed twice with PBS and the supernatant was discarded after centrifugation at 800 × g for 3 min. The cells were mixed homogeneously with ultrapure water. The cell homogenates were transferred to EP tubes and heated in a water bath (100°C, 10 min). The content of ATP was determined according to the instructions of the ATP test kit.

MDA-MB-231 cells were transfected with miFTO inhibitor or inhibitor control for 48 h. The activity of hexokinase and pyruvate kinase were detected according to the instructions of the hexokinase and pyruvate kinase test kits.

After 48 h culture, culture medium from transfected MDA-MB-231 cells was discarded, cells were washed with PBS and lysed on ice for 30 min. Protein extracts were mixed with loading buffer, and boiled at 100°C for 5 min. A total volume of 50 µl of the denatured protein samples were loaded on gels (12% separation gel and 5% spacer gel). A voltage of 80 V was applied to samples and then adjusted to 120 V when protein reached the separation gel. When bromophenol blue entered the separation gel, electrophoresis was stopped. Protein was transferred to PVDF membranes overnight at 4°C. PVDF membranes were washed with TBST 3 times, and blocked with skim milk for 2 h at 37°C. Membranes were treated with primary antibody overnight at 4°C, washed with TBST, and incubated with secondary antibody for 1.5 h at room temperature. Membranes were washed and developed. Protein expression was analyzed with the Odyssey scanning system (LI-COR, Inc., Lincoln, NE, USA). Primary rabbit polyclonal AKT antibody (dilution, 1:500; cat. no. ab38449); rabbit monoclonal p-AKT antibody (dilution, 1:500; cat. no. ab81283), rabbit monoclonal PI3K antibody (dilution, 1:500; cat. no. ab86714), rabbit polyclonalp-PI3K antibody (dilution, 1:500; cat. no. ab182651) and secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2,000; cat. no. ab6721) were all purchased from Abcam (Cambridge, MA, USA).

Data were analyzed with SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). Comparisons between groups were by t-test. P<0.05 was considered to indicate a statistically significant difference.

In the logarithmic growth phase, MCF-7, MDA-MB-231 and HCC1937 cells were harvested for extraction of total RNA. The expression levels of FTO in the 3 groups of cells were detected by real-time quantitative PCR. The relative expression levels of FTO mRNA in MCF-7 cells was 26.89±2.31, 36.23±2.91 in MDA-MB-231 cells and 8.96±3.01 in HCC1937 cells. The levels of FTO mRNA in MCF-7 and MDA-MB-231 cells were significantly higher than in HCC1937 cells (P<0.01) (Fig. 1).

The content of lactic acid and ATP in MDA-MB-231 cells transfected with the miFTO inhibitor or inhibitor control were detected according to the lactic acid test kit and ATP test kit. After 48 h culture, the lactic acid content of the miFTO inhibitor group was 8.97±0.25 mmol/l, the lactic acid content of the inhibitor control group was 17.11±1.02 mmol/l, and the lactic acid content of the blank control group was 17.08±1.32 mmol/l. The lactic acid content of breast cancer cells transfected with miFTO inhibitor was significantly lower compared with the control group and inhibitor control group. FTO mRNA inhibitors can inhibit the production of lactic acid in breast cancer cells (Fig. 2A). After 48 h culture, the ATP content of the miFTO inhibitor group was 31.45±1.58 mmol/l, the ATP content of the inhibitor control group was 44.12±3.12 mmol/l, and the ATP content of the blank control group was 44.56±2.45 mmol/l. The ATP content of breast cancer cells transfected with the miFTO inhibitor was significantly lower compared with the control group and inhibitor control group (P<0.05). FTO mRNA inhibitors can inhibit the production of ATP in breast cancer cells (Fig. 2B).

The activity of hexokinase and pyruvate kinase in MDA-MB-231 cells transfected with miFTO inhibitor or inhibitor control were detected according to the instructions of the hexokinase and pyruvate kinase test kits. The results showed that pyruvate kinase activity in breast cancer cells transfected with miFTO inhibitor and inhibitor control were 0.39±0.01 and 0.68±0.02, respectively, and 0.71±0.03 in the nontransfected cells. Pyruvate kinase activity of breast cancer cells transfected with miFTO inhibitor was significantly lower compared with the control group and inhibitor control group (P<0.01). Hexokinase activity in breast cancer cells transfected with the miFTO inhibitor and inhibitor control were 2.54±0.21 and 4.86±0.25, respectively, and 4.84±0.20 in the nontransfected cells. Hexokinase activity of breast cancer cells transfected with the miFTO inhibitor was significantly lower compared with the control group and inhibitor control group (P<0.01). Therefore, miFTO inhibitors can reduce the activity of hexokinase and pyruvate kinase in breast cancer cells (Fig. 3).

MDA-MB-231 cells transfected with the miFTO inhibitor or inhibitor control for 48 h were harvested, and lysates were used to analyze the expressions of phosphatidylinositol 3-kinase (PI3K), p-PI3K, protein kinase B (Akt), and p-AKT by western blot analysis. After transfection with the miFTO inhibitor, the expressions of PI3K, p-PI3K, AKT and p-AKT were significantly lower compared with the control group and inhibitor control group. The phosphorylated forms of PI3K and AKT decreased significantly (Fig. 4).