Chemicals and reagents
The cervical epithelial SiHa cells line were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s Modified Eagle’s Medium (DMEM) powder, fetal bovine serum (FBS) and L-glutamine were obtained from the Gibco (Grand Island, NY, USA); The Lipofectamine® 2000 transfection reagent was purchased from Santa Cruz (Santa Cruz, California, USA). Dimethyl sulfoxide (DMSO) and Lipopolysaccharide (LPS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). FoxO1 small-interfering RNA (siRNA) were designed and synthesized by Wuhan Genesil Biotechnology Co. Ltd (Wuhan, China). FoxO1 and actin gene primers were designed and synthesized by Shanghai Boya Biotechnology Co. Ltd (Shanghai, China).
SiHa cells were grown in DMEM medium containing with 1% nonessential amino acids, 2 mM of glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% FBS. SiHa cells were cultured in a 5 % CO2 incubator at 37°C to 80-90 % confluence, and then treated with 40 µg/ml LPS for 24 h or other reagents at the indicated times and concentrations. The choice of LPS concentrations was based on previous reports .
Cloning and transfection of FoxO1 vector plasmids
The pcDNA 3.1 expression plasmid (Invitrogen, Carlsbad, CA) expressing pcDNA3.1-FoxO1 vector was created at Hangzhou Hibio Bio-tech Co., Ltd. (Hangzhou, Zhejiang, China). The pcDNA3.1 empty vector was chosen as a negative control. FoxO1: Primer-F: 5′-GCG GGC TGG AAG AAT TCA AT -3′ and Primer-R: 5′- TCC AGT TCC TTC ATT CTG CA-3′. The PCR product was digested with BamHI and EcoRI and ligated into pcDNA 3.1 expression plasmid. The resulting pcDNA3.1-FoxO1 vector or pcDNA3.1 empty vector was then transfected into SiHa cells using Lipofectamine 2000 according to the manufacturer’s instructions. Briefly, before transfection, the SiHa cells were serum starved for 24 h, 500 pmol of pcDNA3.1-FoxO1 vector and 10 μL of Lipofectamine 2000 were diluted in 750 μL of OptiMEM (Life Technologies, Gaithersburg, MD, USA). The solution was pre-incubated for 45 min at 37 °C, then overlaid onto the SiHa cells for 2 h, finally, 2 ml of growth medium (20% foetal bovine serum) were added for further cultured.
FoxO1 siRNA-expressing plasmid construction
The FoxO1 siRNA expression plasmid was constructed using the primers 5′-CCC AAG GCT TTG GTC CTA TC-3′ (forward), 5′-GCC GGA TTC ACT GTA TTC TTG-3′ (reverse). Negative siRNAs were sequenced (An unrelated gene) as follows: 5′- GUA CCG CAC GUC AUU CGU AUC-3′ (forward), 5′-UAC GAA UGA CGU GCG GUA CGU-3′ (reverse). The reconstituted FoxO1 siRNA expression plasmid transfected into SiHa cells, along with LPS (40 µg/ ml). The SiHa cell mitochondrial function and biological function was measured.
Real-time quantitative polymerase chain reaction (real-time qPCR)
Extraction of total RNA from SiHa cells was performed using RNAiso Plus (TakaRa, China). Then, 10 µL of total RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript cDNA Synthesis Kit (Takara, Japan). The following sequences were used to detect FoxO1 mRNA level: Primer-F: 5′-GCG GGC TGG AAG AAT TCA AT -3′ and Primer-R: 5′-TCC AGT TCC TTC ATT CTG CA-3′. β-actin sense: 5′-CGA GCG GGA AAT CGT GCG TGA CAT -3′; and antisense, 5′-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT -3′. Real-time qPCR reactions were performed using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The relative level of FoxO1 mRNA was calculated using the threshold cycle (2-ΔΔCT) method .
Western blot analysis
The SiHa cells were thawed in lysis buffer containing containing 0.5% Deoxycholate, 50mM Tris–HCl (pH 7.0), 0.1% Triton X-100, 150mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 1mM ethylenediaminetetraacetic acid (EDTA). Total proteins were electrophoresed using a 10-15% gradient SDS--polyacrylamide gel and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were then blocked in 5% non-fat milk in PBST for 1 h, and then incubated with primary antibodies specific to FoxO1 (1: 1000 dilution, Abcam: ab52587), Caspase-1 (1: 500 dilution, Santa Cruz, CA, USA) and actin (1: 2000; ab8227, Abcam). The membrane were washed and then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 4000). Visualization of protein band was quantiﬁed using the Enhanced Chemiluminescence Western Detection System (Cell Signaling Technology, Beverly, MA, USA).
The SiHa cells were digested by trypsin and pelleted at 1000 X for 30 min at 4 °C. The mass were cut into 5 µm slice, then treated with 4% H2O2 for 30 min. The antigen retrieval was underwent in 0.01 M citrate buffer (pH 6.0) for 25 min. The slides were incubated with FoxO1 antibodies (dilution, 1: 100, Santa Cruz Biotechnology) for 30 min at 37˚C and 3, 3'‑diaminobenzidine Sigma‑D8001 staining kit (Sigma Aldrich; Merck KGaA) staining. Positive (brown) staining indicates the presence of the HAX‑1 protein, as detected by light microscopy (magnification, x200).
The production of IL-1β was detected by ELISA kit (R&D System, Minneapolis, MN). 200 µL of supernatant was added to each well and incubated for 45 min at 37 °C. After incubation, the plate was washed four times, and was then added with 200 μl of conjugate for 45 min at 37 °C. After washing four times, 200 µL substrate solution was added for 20 min in the dark. The absorbance was read using an ELISA Reader at 450 nm after the addition of 2 M sulfuric acid. The level of IL-1β in culture media was calculated according to a standard curve.
The cultured SiHa cells were digested by trypsin and pelleted at 12000 × g for 10 min at 4 °C. The cells mass were fixed with 2 % paraformaldehyde and placed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Then the pellet was washed twice and dehydrated in a gradient series of ethanol solutions (25%, 50%, 70%, 80%, 90% and twice 100% for 15 min). The sample was treated with acetone and ﬂat-embedded in Durcupan (Fluka Chemic AG, Buchs, Switzerland), and then sectioned to 60-70 nm thickness on 300 mesh copper slot grids. The ultrastructure of cells was examined at 5200 X magniﬁcation (Observation of mitochondrial morphology) and 2500 X magniﬁcation (Observation of cell apoptosis morphology), and the images were viewed under a Zeiss 109 electron microscope (Carl Zeiss, Oberkochen, Germany).
Assay of intracellular ROS
The production of intracellular ROS was measured by a H2DCFDA based ROS assay kit (Beyotime, Shanghai, China). Briefly, the SiHa cells were applied with different treatments, cells were harvested, and then incubated with 10 µM final concentration of H2DCFDA for 30 min in the dark at 37˚C. Flow cytometry (BD FACSCalibur, San Jose, CA, USA) was used to detect the intracellular ROS generation with excitation wavelength and the emission wavelength at 488 nm and 530 nm respectively.
Measurement of mitochondrial membrane potential (ΔΨm)
Loss of mitochondrial membrane potential (ΔΨm) was examined in SiHa cells using the fluorescent cationic dye JC‑1 (Molecular Probes; Thermo Fisher Scientific, Inc.). The SiHa cells were applied with different treatments and then stained with 10 µM JC‑1 for 20 min at RT. The ﬂuorescence intensities with the excitation wavelength and the emission wavelength at 485 nm and 530 nm respectively using fluorescence microscopy (magnification, x200).
SiHa cells migration analysis
The siHa cells migration were detected using the transwell (Corning Incorporated, Corning, NY, USA) assay. The SiHa cells were starved for 24 h and were then harvested. SiHa cells were resuspended in culture medium at least 2 × 106 cells/ml. The upper chamber was filled with 100 μl cell suspension and the lower chamber was added with 600 μl culture medium containing 20% FBS. The chambers were incubated for 24 h at 37°C. The migration ability of SiHa cells were fixes with 95% ethanol and then stained with 0.1% crystal violet staining. In five different fields per filter, the migrated cells were counted under a microscopic (×400).
SiHa cell proliferation assay
The SiHa cells were starved for 24 h and were then harvested. SiHa cells were resuspended in culture medium at least 2 × 106 cells/ml. 3H‑thymidine were incorporated into SiHa cells to indicate DNA synthesis. The cultured SiHa cells were trypsinized and were then harvested onto a glass fiber filter paper. Scintillation solution was added to detect the radioactivity, which was counted by a TopCount NxT scintillation counter (LKB Instruments, Mount Waverly, Victoria, Australia).
All data are presented as mean ± standard deviation (SD). Student’s t test was used to compare the means of two groups. p-values less than 0.05 were considered significant (*p < 0.05; ** p < 0.01; # p > 0.05). Statistical significance was calculated using SPSS18.0. All experiments were performed at least three independent experiments.