Tissue samples
Between 2008 and 2014, clinical data and cervical SCC samples from patients were collected at the First Affiliated Hospital and the Third Affiliated Hospital of the Medical College of Shihezi University in Xinjiang, China, with the approval of the ethical committee of each hospital. Written informed consent was obtained from patients. Patients received neither chemotherapy nor radiotherapy before sample collection. The diagnoses were confirmed independently by two experienced pathologists. Cervical SCC tissues and adjacent normal cervical tissues were collected from each patient. Tissue samples were frozen immediately after removal and stored at − 80 °C.
Immunohistochemical staining
Tumor tissues were fixed in 10% formalin, embedded in paraffin, and cut into 4 μm-thick sections. For immunohistochemical staining, tissue sections were deparaffinized in xylene and rehydrated in descending grades of ethanol. Endogenous peroxidase activity was blocked with methanol containing 0.3% H2O2 for 30 min and then washed in PBS. Antigen retrieval was performed by microwaving with citrate phosphate buffer (pH 6.0). The sections were then placed with the primary antibodies at 4 °C overnight. After incubation, the sections were washed in PBS for 3 min. The sections were then washed five times with PBS for several seconds, incubated with secondary antibodies at 37 °C for 30 min, and washed twice with PBS. After incubation with the secondary antibodies, staining was completed using anti-mouse peroxidase and DAB substrate. Tissue sections were counterstained with hematoxylin. IFITM1, Ki-67, and PCNA protein signals were scored on the following scale considering both the proportion of cells stained and the intensity of staining in those cells: score 0, no cells stained; score 1, weak or absent nuclear staining and < 5% of cells stained; score 2, nuclear staining and between 5 and 25% of the cells stained; score 3, nuclear staining and between 26 and 50% of the cells stained; and score 4, nuclear staining and more than 50% of the cells stained. Two observers scored independently using this scale.
Real-time RT-PCR
Total RNA was extracted from cell or tissue samples using TRIzol reagent according to the manufacturer’s protocol (Invitrogen). The RNA concentration was determined by agarose gel electrophoresis or absorbance at 260 nm. cDNA was synthesized with Invitrogen’s SuperScript One-Step RT-PCR Kit; each reaction contained 2 μg of total RNA, 2 μL of Oligo(dT) (500 μg/mL), and 7.5 μL of DEPC water. Reactions were heated for denaturation at 65 °C for 5 min and then quenched on ice for 5 min. The following reagents were added to each reaction: 4 µL of 5 × first buffer, 2 µL of 0.1 M DTT, 1 µL of dNTPs (10 mM each), 0.5 μL of RNase inhibitor (40 U/μL), and 1 μL of M-MLV (200 U/μL); the total volume of each reaction was 20 μL. The reactions were maintained at 25 °C for 10 min and 37 °C for 1 h. The reactions were terminated at 70 °C for 10 min. IFITM1 mRNA quantitative PCR amplification was performed on Light Cycler 480 (Roche Diagnostics) with the forward primer 5′-ACTCAACACTTCCTTCCCCAA-3′ and the reverse primer 5′-CTTCCTGTCCCTAGACTTCACG-3′. The amplicons were 231 bp in size. To normalize the amount of cDNA in each sample, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified in the control experiment with specific primers (forward: 5′-TGTTGCCATCAATGACCCCTT-3′; reverse: 5′-CTCCACGACGTACTCAGCG-3′); the amplicons were 202 bp in size. PCR reactions were performed in a volume of 20 μL, consisting of 10 μL of 2 × PCR buffer, 500 ng of cDNA, 10 μL of 2 × PCR buffer for EvaGreen, 0.6 μL of 20 × EvaGreen, 0.6 μL of forward primer and reverse primer (10 μM) each, and 0.3 μL of Cap Taq polymerase (5 U/μL). DEPC water was added to bring the volume to 20 μL. Reaction conditions were as follows: initial denaturation for 5 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C, primer annealing for 15 s at 55 °C, extension for 20 s at 72 °C, and UPL fluorescence measurement for 3 s at 76 °C. The GAPDH gene was used as an endogenous control to normalize the difference in the amount of cDNA in each sample.
DNA preparation and detection of HPV
DNA was extracted with an SK1252 genomic DNA isolation mini kit (Shanghai Sangon Biological Engineering Technology and Services Company) according to the manufacturer’s protocol. HPV detection and typing were performed in all samples; HPV16 and HPV18 were analyzed by PCR. The primers used were pHPV16-F (5′-GACCCAGAAAGTTACCACAG-3′) with pHPV16-R (5′-CACAACGGTTTGTTGTATTG-3′) for HPV16 virus detection, as well as pHPV18-F (5′-TGCCAGAAACCGTTGAATCC-3′) with pHPV18-R (5′-TCTGAGTCGCTTAATTGCTC-3′) for HPV18 virus detection. Both HPV16 and HPV18 PCR amplicons were 268 bp in size.
DNA modification by bisulfite treatment and methylation-specific PCR (MSP)
The DNA samples were subjected to bisulfite treatment using CpGenome™ DNA modification kit S7820 (CHEMICON, Temecula, CA, USA) according to the manufacturer’s protocol. The modified DNA was purified, followed by ethanol precipitation and then stored at − 80 °C. Methylated primers were pM-IFITM1-F (5′-GAGATTTTCGTGTTCGATTATGTC-3′) and pM-IFITM1-R (5′-ATAAAACCCCAAACTCACCG-3′), whereas unmethylated primers were pUM-IFITM1-F (5′-AGATTTTTGTGTTTGATTATGTTGT-3′) and pUM-IFITM1-R (5′-ATAAAACCCCAAACTCACCAAC-3′). Each reaction was 25 μL containing 1 μL of modified DNA template, 0.5 μL of forward and reverse primer each (0.5 μmol/L), 12.5 μL of Taq DNA Polymerase Mix, and 10.5 μL of H2O. The reaction conditions were as follows: 34 cycles at 95 °C for 5 min, 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, and an extension step at 72 °C for 10 min. PCR results were checked by agarose gel (2%) electrophoresis.
RT-PCR
mRNA expression in the 10 methylated and 10 unmethylated cervical tissues was measured by RT-PCR. Total RNA was extracted with TRIzol Reagent (Life Technologies) according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using an oligo(dt) primer and reverse transcriptase (Fermentas). About 2 μL of cDNA products was then PCR-amplified using IFITM1 gene primers and Taq DNA Polymerase Mix according to the manufacturer’s instructions. RT-PCR primers were pRT-IFITM1-F (forward: 5′-ATGTCGTCTGGTCCCTGTTC-3′) and pRT-IFITM1-R (reverse: 5′-GTCATGAGGATGCCCAGAAT-3′). GAPDH served as the internal control; the primers were GAPDH-F (forward, 5′-GCCAAAAGGGTCATCATCTC-3′) and GAPDH-R (reverse, 5′-GTAGAGGCAGGGATGATGTTC-3′). 5 μL of PCR products was run on 1% agarose gel to determine IFITM1 gene expression.
Cell culture and transfection
cDNA of IFITM1 gene was cloned into pcDNA3.1(−) expression vector and confirmed by gene sequencing. In addition, pcDNA3.1(−) vectors were used as the control. HeLa cells were plated at 2 × 105 cells per well in a six-well cell culture plate at 24 h before transfection. Subsequently, 2 μg of IFITM1 constructs and the control vector were mixed with 6 μL of Lipofectamine 2000 (Invitrogen). The mixture was incubated at room temperature for 10 min. After washing the cells with 1 × PBS, the DNA/Superfect mixtures were transferred to HeLa cells. The control pcDNA3.1 vector was transferred into HeLa cells separately. Transfected HeLa cells were then incubated at 37 °C in 5% CO2 for 24 h. The effect of IFITM1 overexpression was determined by measuring immunofluorescence luciferase activity using an assay system according to the manufacturer’s protocol (Promega). Each experiment was repeated with multiple batches of cells.
Wound healing assay
Cells were seeded onto six-well plates (1 × 105 cells/dish). When cells reached over 90% confluence, a scratch was made across the cell monolayer with a tip. Cells were gently washed with PBS three times and maintained in a fresh medium. Cells were incubated for 24 h and photographed using an inverted tissue culture microscope at 100 × magnification. Assays were performed at least three times, and data are presented as the mean ± SD. The migration potential between IFITM1 expressing cells and control cells was compared by relative gap distance. P < 0.05 was considered statistically significant.
Cell migration and invasion assays
Cells were serum-starved overnight. The top chambers of 6.5 mm Corning Costar transwells (Corning, USA) were loaded with 0.2 mL of cells (5 × 105 cells/mL) in serum-free media. Complete media (0.6 mL) were added to the bottom wells, and cells were incubated at 37 °C overnight. Cells that migrated through the membrane were fixed, stained with 0.1% crystal violet, and examined under a light microscope. Images of the cells at the bottom of the membrane were captured using a Canon camera and a Zeiss microscope. The mean values were obtained from three individual experiments using Student’s t tests. For cell invasion assay, cells were serum-starved overnight. The 24-well cell culture inserts (8 mm pore size, BD Biosciences, San Jose, USA) were loaded with 0.5 mL of cells (5 × 105 cells/mL) in serum-free media. Complete media (0.5 mL) were added to the bottom wells, and cells were incubated at 37 °C for 24 h. Cells were fixed, stained, and analyzed as described above.
Cell proliferation determined by cell counting kit-8 assay
Proliferation of HeLa cells transfected by the IFITM1 pcDNA3.1 construct and pcDNA3.1 was determined using cell counting kit-8 reagents (Dojindo Laboratories, Japan). HeLa cells were seeded in 96-well plates (Falcon; Becton–Dickinson Labware) at 2 × 105 cells per well in DMEM containing 10% FBS. The cells were incubated for 24, 48, 72 h at 37 °C. Spectrometric absorbance at 450 nm was measured with a microplate reader (X-Mark; Bio-Rad, USA); the quantification was repeated five times.
Cell cycle analysis
IFITM1—pcDNA3.1 construct- or pcDNA3.1 vector-transfected cells were digested by trypsin, fixed with 70% ethanol, and stored at 4 °C until analysis. The cells were suspended in 100 μL of 180 μg/mL RNA enzyme A in and then incubated for 30 min at room temperature. Propidium iodide (PI) solution (50 μg/mL; Merck, Darmstadt, Germany) was added to the cells. The cells were then kept in a dark room for 15 min. The DNA content of cells was measured by flow cytometry (FACScan system, Becton–Dickinson). The G0/G1 phase ratio, as well as S and G2/M phase ratio, was determined from flow cytometry data.
Cell apoptosis detected by flow cytometry
Apoptosis was analyzed by Annexin V-fluorescein isothiocyanate staining using an Alexa Fluor 488 Annexin V kit (Invitrogen). In total, 1.0 × 106 of IFITM1—pcDNA3.1 construct-transfected cells, as well as the same amount of pcDNA3.1 vector-transfected cells, were washed twice with cold PBS. Cells were then resuspended in 1 × Annexin-binding buffer for 30 min. Cell apoptosis was determined by Annexin V-fluorescein isothiocyanate/PI double staining. Cell transfection was analyzed on FACS calibration with the software Cell Quest (Becton–Dickinson). All experiments were carried out in triplicate.
Statistical analysis
SPSS17 software was used in all statistical analyses. IFITM1 protein levels were compared between cervical carcinoma and chronic cervicitis using non-parametric test. Results of the triplicates were represented as the mean ± standard deviation (if applicable). Results were considered statistically significant if P < 0.05 in two-tailed Student’s t tests.