Bioinformatics analysis
The Cancer Genome Atlas (TCGA, http//gdc.cancer.gov/) database including 338 OSCC tumor tissues and 32 normal tissues was used to detect ALG3 expression in OSCC, and evaluate the relationship between ALG3 expression and patients overall survival. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to detect the pathway closely related to ALG3 in OSCC. Clinical correlation analysis was implemented to assess the association between ALG3 expression and clinical parameters.
Cell culture and gene expression
Cell Bank of the Chinese Academy of Sciences (Shanghai, China) provided the cell lines including OSCC cell lines (CAL27 and TCA-83) and normal cell line HOEC. All cells were sustained in Roswell Park Memorial Institute (RPMI) 1640 medium, which accompanied by 10% fetal bovine serum (FBS), 100U/mL penicillin, and 0.1 mg/mL streptomycin, under an atmosphere including 5% CO2 at 37 oC.
ALG3 over-expression or knockdown assays were carried out by using pcDNA3.1-ALG3 and si-ALG3. Sequences of si-ALG3 and si-negative control (NC) were exhibited as follows: si-NC: 5’-UUCUUCGAAGGUGUCACGUTT-3’, si-ALG3#1: 5’-GGUUUCGUGUACAUCUUUAUG-3’, si-ALG3#2: 5’- GGACCUGAGUCUACCCUCAGG-3’. Si-NC, si-ALG3#1, si-ALG3#2, pcDNA3.1-vector, pcDNA3.1-ALG3 were brought from Sangon (Shanghai, China). When the confluence of cells in the six-well plates reached 80%, Lipofectamine2000 was applied to mediate cell transfection. After cultivation for 24 h, the expression of ALG3 could be observed.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Trizol reagent was applied to isolate the total RNA according to the supplier’s recommendations. Equal amount of RNA was utilized to synthesize cDNA by PrimeScriptTMRT reagent Kit (Takara, Dalian, China). cDNA abundance was detected by qRT-PCR with SYBR Premix EX Taq (Takara, Dalian, China). 2-△△Ct method was used to determine the relative expression of ALG3. GAPDH was regarded as an internal standard for ALG3 detection. The following primers were used:
ALG3: F-5’-CTTTGCTGTGCTCTACCTGGCT-3’,
R: 5’-CGCAGCACAAAGATGGAGTGGA-3’;
GAPDH: F: 5’-GTCTCCTCTGACTTCAACAGCG-3’,
R: 5’-ACCACCCTGTTGCTGTAGCCAA-3’.
Western blotting
Proteins samples from CAL27 and TCA-83 cells were lysed in RIPA buffer (Beyotime, Shanghai, China) on ice. A total of 20 µg proteins were isolated on 12% sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto membranes. After blocking with 5% non-fat milk for 2 h at ambient temperature, the membranes were incubated with the primary antibodies against the target proteins (Abcam, Cambridge, UK) including ALG3, MCM7, CCNB2, CDK1, PCNA and GAPDH at 4 oC overnight. Thereafter, the membranes were rinsed with tris buffered saline tween (TBST) for three times and then incubated with indicated secondary antibodies. Finally, chemiluminesence analysis was implemented to analyze the signals with the support of the enhanced chemilunimescence (ECL).
Cell proliferation examination
Cell proliferation was evaluated with the assistance of cell counting kit-8 (CCK-8, Beyotime, Shanghai, China) in steps with the supplier’s specification. Firstly, a total of 1000 CAL27 or TCA-83 cells were implanted in triplicate into 96-well plates and cultured in 100 µL RPMI-1640 medium. The optical density (OD) values of the cells were measured every 24 h. Before the measurement, 10 µL CCK8 solution was put into the medium and incubated for 2 h. The OD values were measured under the absorbance of 450 nm by using a microplate reader. Finally, a growth curve was made by using Graphpad Prism 6.0.
Plate clone formation assay
Firstly, 3mL of complete medium including 200 cells was added into a well from a 6-well plate and cultured at 37 °C and 5% CO2 incubator for about 10-14 days. Afterwards, cells were lightly rinsed and stained. Finally, the colonies were counted and pictured.
Detection of cell invasion and migration abilities
Transwell chambers (8 µm pore size, Corning Costar, Cambridge) were used to detect the invasion and migration abilities of CAL27 or TCA-83 cells. For invasion experiment, 1×105 cells suspended in 200 µL serum-free RMPI-1640 medium were seeded in triplicate into the upper chamber of 24-well chambers. The under compartment included 500 µL RMPI-1640 medium with 10% FBS. After cultivation in 37 °C incubator for 24 h, the chambers were rinsed by phosphate buffer solution (PBS) gently to remove the cells gluing to the upper surface. After being fixed and stained, the cells were photographed under a microscope.
The procedure of the migration experiment was consistent with that of the invasion experiment, except that the upper chamber did not need to be pre-coated.
Data analysis
The values in this research were exhibited as mean ± standard deviation (SD). Statistical analysis was implemented by SPSS22.0 and Graphpad Prism 6.0. The comparisons between the experimental group and control group were evaluated by student’s t test and one way ANOVA. The overall survival of OSCC patients with different expression of ALG3 was analyzed by Kaplan-Meier method with the log-rank test. Chi-square analysis was used to assess the association between the ALG3 expression and the clinicopathological parameter of OSCC. A P value greater than 0.05 was considered statistically insignificant.