Cell culture and chemicals
Cal27 and HSC-2 were obtained from State Key Laboratory of Oral Diseases, Sichuan University, China. Cells were cultured in high glucose DMEM (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin and streptomycin (100 μg/ml) (Hyclone, USA). Metformin was purchased from Sigma-Aldrich (USA).
Cell proliferation assay
Cal27 and HSC-2 cells (3 × 103) were seeded into 96-well plates and treated with 0, 2.5, 5, 10, 20 and 40 mM metformin for 24, 48, 72 and 96 h. The cells were incubated with Cell counting kit-8 (CCK8; Donjido, Japan) at 37 ℃ for 1.5 h following the manufacturer’s instruction. The absorbance at 450 nm was detected by a microplate reader (Thermo, USA).
Cells (500 cells per well) were seeded into 6-well plates and incubated overnight allowing to cell adherence. Then cells were treated with 0, 5 and 10mM metformin for 14 days until the visible colonies were generated. After the fixation by 4% paraformaldehyde, colonies were stained with crystal violet for 5 min. The colonies were photographed by camera and counted under the phase-contrast microscope.
Cell cycle assay
Cal27 and HSC-2 cells (3 × 105) were seeded into the 6-well plates and treated with 10, 20 and 40 mM metformin for 48 h. Then cells were fixed with 75% ethanol at 4 ℃ for 4 h and the cell cycle was detected by using PI kit (KGA512, Jikai, China) following the recommended instruction.
Wound healing assay
Cal27 and HSC-2 cells (8 × 105) were seeded into 6-well plates and wounded by pipette tips after incubation overnight. Then cells were cultured with serum-free medium for 24 h. The images were captured using phase-contrast microscopy at 0 h and 24 h.
Cal27 cells (5×106) in logarithm stage were suspended in 200 μl phosphate-buffered saline (PBS) and injected into the right armpit of 6-week-old BALB/C nude mice. When the tumors were palpable one week later after the injection, mice were randomly divided into the group treated with metformin and the other without metformin. For the treated group, metformin (200 μg/ml) was added into the drink water in a light-protected bottle and the control group were supplied with normal drink water. The animal weight, blood glucose and tumor volume were detected until the tumor volume was up to 1,000 mm3. The tumor volume was calculated according to the formula: volume = (major diameter2×minor diameter) / 2. The mice were sacrificed after 3-week treatment and tissues were fixed with 4% paraformaldehyde for hematoxylin-eosin (HE) stain and immunohistochemistry (IHC) analysis.
The IHC analysis was conducted according to the instruction of antibodies’ manufacturers. The primary antibodies were as following: H3K27me3 (#9733, Cell Signaling Technology, USA) and H3K27ac (#8173, Cell Signaling Technology, USA).
OSCC samples collection
The OSCC samples and the corresponding adjacent normal tissues were obtained from the inpatients at West China School of Stomatology, Sichuan University. There were 2 patients with squamous cell carcinoma (SCC) on tongue, 2 on oral floor, and 1 on buccal. All patients were diagnosed as primary SCC and conducted with radical surgical resection. Informed consents of all patients were obtained.
Cells and tissues were lysed in RIPA (P0013B, Beyotime, China) supplemented with 1mM PMSF (ST506, Beyotime, China) and 0.01mM phosphatase inhibitors (P1260, Solarbio, China). Samples with 20 mg protein in each group was separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). The primary antibodies were used as following: β-actin (1:2000, 20536-1-AP, Protein tech, China), EZH2 (1:1000, #5246, Cell Signaling Technology, USA), SUZ12 (1:1000, #3737, Cell signaling Technology, USA), EED (1:1000, 16818-1-AP, Protein tech, China), Histone 3 (1:2000, 17168-1-AP Protein tech, China), H3K27me3 (1:1000, #9733, Cell Signaling Technology, USA), and H3K27ac (1:1000, #8173, Cell Signaling Technology, USA). After incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, #7074, Cell Signaling Technology, USA), the blots were visualized with chemiluminescent substrate (US Everbright Inc., China) and quantified by Image Lab (Bio-Rad, USA).
Endofectin Max (Genecoopia) was used for siRNA transfection following the manufacturer’s recommendation. Cells were transfected with the mixture of opti-MEM (Gibco, USA), endofectin and siRNA. Control siRNA was used as a negative group. After incubation for 48 h, cells were treated with 10mM metformin for 48 h. SiRNA was designed and purchased from Genepharma (China). The sequence of siRNA-EZH2 were GCAGCUUUCUGUUCAACUUTT, AAGUUGAACAGAAAGCUGCTT. The sequence of siRNA-Control was UUCUCCGAACGUGUCACGUTT, ACGUGACACGUUCGGAGAATT.
OSCC cells, Cal27 and HSC-2, were treated with or without 10mM metformin for 6 days. According to the methods in previous study, cells ( 1×106 ) were lysed with Trizol reagent (Invitrogen) and oligo (dT) magnetic beads (Thermo, USA) were used to enrich mRNA. After mRNA was divided into small fragments, cDNA was synergized and purified with magnetic beads. The sequencing was carried out on Illumina Novaseq 6000 in Novogene Bioinformatics Technology (China). Control group consisted of Cal27 and HSC-2 treated without metformin and case group consisted of Cal27 and HSC-2 treated with 10 mM metformin. The data were analyzed by Novogene Bioinformatics Technology (China). Each group consisted of two replicates from Cal27 and HSC-2.
OSCC cells, Cal27 and HSC-2, were cultured with or without 10 mM metformin for 6 days. Then 1×108 cells were cross-linked using 1% paraformaldehyde for 5 min and quenched with 125mM glycine at room temperature. DNA was purified by associating with the antibodies of H3K27me3 (#9733, Cell Signaling Technology, USA), H3K27ac (#8173, Cell Signaling Technology, USA) and Normal Rabbit IgG (#2729, Cell Signaling Technology, USA) as previously described. The DNA fragments were end-paired 5’-phosphorylated, 3’-dA-tailed and ligated to adapters using the Acegen DNA library Prep kit (Acegen, AG0810). The adapter-ligated DNAs were purified and amplified by 12 cycles of PCR using illumine 8-bp dual index primers. The constructed libraries were then analyzed by Agilent 2100 bioanalyzer and sequenced on Illumina Novaseq 6000 using a 150 × 2 paired-end sequencing protocol. The sequencing and data analysis were conducted in Novogene Bioinformatics Technology. Each group consisted of two replicates from Cal27 and HSC-2.
All data are presented as mean ± SD. All experiments were conducted three times independently except for extra interpretation. One-way analysis of variance (ANOVA) was used to compare the difference in multiple group and Student’s t test was used to analyze the difference of two groups. P<0.05 was set to be statistical significance.