Cell lines and cell culture
Human oral cancer cell lines HN30 and CAL27 were bought from Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM medium (Gibco, CA, USA) containing 10% FBS (Gibco, CA, USA) and 1% penicillin-streptomycin (Beyotime, Shanghai, China).
Synthesis of small interfere RNA oligonucleotides and transfection
Two small RNA oligonucleotide fragments (siRNA-1#, siRNA-2#) targeting human ANLN gene were designed and synthesized. The target sequence for human ANLN gene was listed in Table 1. Then Lipo3000 reagent was used to transfer the two siRNA into oral cancer cells. In brief, 1μL of Lipo3000 (Invitrogen, CA, USA) was mixed with 50 pmol siRNA for 20 min at room temperature. Then the mix was added into prepared cells in 24-well plates. After culture for 6 h, the supernatant was removed and new fresh medium was added. Then cells were cultured for another 48 h.
Realtime quantitative polymerase chain reaction assay (qPCR)
Total RNA was extracted from cultured cells with RNA easy Kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction. Briefly, cells were lysed with lysis buffer and binding buffer was added into the lysis mix. Then the mixture was transferred into collection column and washed with washing buffer A/B. At last, RNA was eluted with elution buffer and quantified on an ultraviolet spectrophotometer (Onedrop1000, Hangzhou, China).
About 0.5 μg of total RNA was used to synthesize first chain of cDNA with cDNA synthesize kit (Yeasen, Shanghai, China). Then 1 μL cDNA was used for qPCR assay with SYBR GREEN mix (Beyotime, Shanghai, China) on ABI 7000 system (Applied Biosystems, USA). The protocol for qPCR assay was as following: 95℃, 5 min; (95℃, 10 sec; 60℃, 15 sec) for 40 cycles. b-actin was chosen as internal control. The relative mRNA expression level of each gene was calculated by 2-△△CT method. The primers were showed in Table 2.
Western blotting assay
Total protein from cultured cells was prepared with Protein-extract kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction. In brief, cells were collected by centrifugation at 1200 rpm for 5 min at room temperature. The cell body was resuspended with lysis buffer and transferred into the protein column and washed with serial buffer. At last, protein was eluted with elution buffer and the concentration of total protein was detected on an ultraviolet spectrophotometer (Onedrop1000, Hangzhou, China).
10 μg total protein was separated on 15% SDS-PAGE GEL for 1.5 h. Then candidate protein was transferred onto PVDF membranes (Beyotime, Shanghai, China) and analyzed by western blotting analysis. In brief, the PVDF membranes were blocked with 1% BSA for 1 h at room temperature and washed with PBS for three times. Then, primary antibody against each candidate protein was co-cultured with PVDF membrane for 12 h at 4℃. After washed with PBS for three times, secondary antibody was co-cultured with the PVDF membranes for 2 h at room temperature. At last, after the PVDF membranes were washed with PBS, each candidate protein was detected by ECL kit (Yeasen, Shanghai, China) according to the manufacturer’s instructions. The information of primary antibodies were as following: PI3K (ab140307, 1:2000, Abcam, CA, USA), mTOR (ab134903, 1:6000, Abcam, CA, USA), AKT (ab18785, 1:1000, Abcam, CA, USA), PDK-1 (ab207450, 1:2000, Abcam, CA, USA), GAPDH (ab9485, 1:2000, Abcam, CA, USA).
Cell proliferation assay
Oral cancer cells treated with siRNA were seeded into a 96-well plate at 5×103/100μL/well and cultured for 96 h. Then 10% of total volume of CCK-8 reagent was added into each well every 24 h and cultured for another 1 h. The absorbance value at 450 nm wavelength was detected on a microplate reader (Tecan, Switzerland). Each experiment was repeated independently for three times.
Plate colony formation
Oral cancer cells treated with siRNA were seeded into a 24-well plate at 0.5×103/100μL/well and cultured for 14 days. After 14 days, cells were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 15 min and washed with PBS for three times. Then cells were stained in 0.5% crystal staining buffer (Sangon, Shanghai, China) for 15 min at room temperature. After washed with PBS for three times, positively stained cells were calculated under a microscope (OLYMPUS, Tokyo, Japan). Each experiment was repeated independently for three times.
Transwell assay
Transwell insert with 8 μm pore was treated with 200 μL matrigel (BD science, USA) and placed overnight. After washed with 200 μL culture medium, oral cancer cells treated with siRNA were seeded into the insert at 1×104/300μL/well and placed into a 24-well plate. Culture medium with no FBS was added into the upper room of the insert when culture medium with 10% FBS was added into the lower well of the 24-well plate. After cultured for 48 h, cells in the upper layer of the insert were scraped gently and cells in the lower layer of the insert were fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 15 min and washed with PBS for three times. Then cells were stained in 0.5% crystal staining buffer (Sangon, Shanghai, China) for 15 min at room temperature. After washed with PBS for three times, positively stained cells were calculated under a microscope (OLYMPUS, Tokyo, Japan). Each experiment was repeated independently for three times.
Cell apoptosis detection
Oral cancer cells treated with siRNA were seeded into a 6-well plate at 2×105/500μL/well and culture for 48 h. Then cells were treated with Apoptosis-detecton-kit (Beyotime, Shanghai, China). In brief, cells were collected by centrifugation at 1200 rpm for 5 min at room temperature. Then cells were resuspended in 100 μL staining buffer containing 5 μL Annexin V/FITC and 10μL PI reagent. After culture for 15 min at room temperature in the dark, 400μL staining buffer was added and cell apoptosis was analyzed on a flow cytometer (BD, FACSCelesta, CA, USA). Each experiment was repeated independently for three times.
Cell cycle detection
Oral cancer cells treated with siRNA were seeded into a 6-well plate at 2×105/500μL/well and culture for 48 h. Then cells were treated with Cell cycle-detecton-kit (Yeasen, Shanghai, China). In brief, cells were collected by centrifugation at 1200 rpm for 5 min at room temperature. Then cells were resuspended in 500 μL staining buffer containing 10 μL RNase A and 10μL PI reagent. After culture for 30 min at room temperature in the dark, cell cycle distribution was analyzed on a flow cytometer (BD, FACSCelesta, CA, USA). Each experiment was repeated independently for three times.
Statistical analysis
All statistical analysis was carried out on SPSS 16.0 software. All data was displayed as mean value plus standard variation (mean±sd). In brief, the difference between two groups was analyzed by unpaired student’t T test. One-way ANOVA analysis was used to analyze the difference among multiple groups. P value less than 0.05 was considered as statistically significant.