TIMER Database
Tumor Immune Estimation Resource (TIMER) database (http://timer.cistrome.org/) was used to explore the transcriptional level of EIF2S3 in different type of cancers. And the correlations between the expression of EIF2S3 and immune cells infiltration including B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages and dendritic cells were analyzed. In addition, the relationship between EIF2S3 expression and immune cell biomarkers was further conducted to illustrate the important role of EIF2S3 in immune infiltration of HCC.
Tcga Database
The Cancer Gene Atlas (TCGA) database (https://cancergenome.nih.gov/) is an online database which includes data from patients with 33 types of cancer. Here, RNA-seq data and clinical information of 374 HCC and 50 normal liver samples were used to analyzed the level of EIF2S3 in HCC. Next, univariate Cox regression analysis and multivariate Cox regression analysis were performed to evaluate the significance of EIF2S3 expression and clinical characteristics in HCC. And, according to the median EIF2S3 expression level, HCC patients were divided into high expression group and low expression group to assess the difference of theses clinical characteristics.
Gepia Database
Gene Expression Profiling Interactive (GEPIA) database (http://gepia2.cancer-pku.cn) which is an online tumor microarray database retrieved from the UCSC Xena server was utilized to analyze the expression levels of EIF2S3 in HCC which do not match the corresponding normal tissues in the TCGA database and the correlation between EIF2S3 expression and immune cell biomarkers.
Kaplan-meier Plotter Analysis
Kaplan-Meier Plotter Analysis(https://kmplot.com/)is an online database for the analysis of cancer patient survival curves, which includes gene expression data and survival information of clinical cancer patients. According to the median EIF2S3 expression level, HCC patients were divided into high expression group and low expression group to evaluate the prognostic value of EIF2S3. We analyzed the relationship between EIF2S3 expression level and overall survival (OS), recurrence-free survival (RFS), progression-free survival (PFS) and disease-specific survival (DSS) of HCC patients though Kaplan-Meier survival curves, hazard ratios (HRs), 95% confidence intervals, and log-rank p values.
Biological Function Analysis
The 50 genes (PCC > 0.56) with the highest and lowest co-expression coefficient with EIF2S3 were identified by GEPIA database, respectively. And, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of these genes in HCC were analyzed by LinkedOmics database.
Cell Culture
HCC cell lines MHCC97H, HCCLM3, Hep3B, Huh-7, and normal liver cell line 7702 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and preserved at the Laboratory of the Molecular Centre of the Second Affiliated Hospital of Nanchang University (Nanchang, China). DMEM medium and Fetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. The cryogenic vials with frozen cells were placed in a water bath for rapid thawing and cells were suspended in DMEM medium supplement with 10% FBS. Then cells were cultured incubated at 37˚C under a humidified atmosphere with 5% CO2. After 12 hours, the morphology and growth density of cells were observed with light microscope. When the density of cells grew to about 90%, the original medium was discarded. After digested with trypsin, cells were collected as cells suspension and centrifuged. The cell precipitates were resuspended and passaged at a ratio of 1:3 for further experiments.
Cell Transfection
In order to validate the role of EIF2S3 in HCC, EIF2S3 siRNA sequences (Ribobio, Guangzhou, China) and NC-siRNA sequences (Ribobio, Guangzhou, China) were designed and synthesized. siEIF2S3-2 sequences were GTCGGAGCTTTACCTGAGA. HCC cells were transfected with 100nM of siRNA, NC-siRNA or the same volume of PBS using riboFECTTM CP Reagent (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. After transfection for 8h in low serum culture condition, cells were cultured for 48h and then conducted functional tests or protein extraction.
Western Blotting
Western blotting
In order to validate EIF2S3 protein expression in different HCC cell lines and the silencing effect of the EIF2S3 interference sequence, total protein was exacted from HCC cells. First, cells were rinsed by PBS and lysed in RIPA buffer with a protease (Beyotime Biotechnology, Shanghai, China). After the protein concentration was identified by BCA kit (Beyotime Biotechnology, Shanghai, China), the extracted protein supernatant was denaturised with loading buffer and then boiled at 100℃ for 10 minutes. Secondly, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocked with 5% non-fat milk for 1 hours, membranes were incubated with primary antibody over night at 4℃. EIF2S3 rabbit antibody (Proteintech Group, Inc, Wuhan, China) was diluted at 1:500 and GAPDH rabbit antibody (Proteintech Group, Inc, Wuhan, China) was diluted at 1:5000. Then, membranes were incubated in the secondary antibody (1:5000, Proteintech Group, Inc, Wuhan, China) at room temperature for 1hour. Finally, membranes were visualized with the ECL detection system and analyzed by Image Lab software (National Institutes of Health).
Cell Viability Assay
According to the operation protocol, CCK-8 kit (MCE, Shanghai, China) was used to assess cell viability. First, MHCC97H and HCCLM3 cells were seeded on 96-well plates at a density of 5000 cells/well. After 24 hours, EIF2S3 siRNA sequences were transfected into cells. Absorbance was recorded at 450 nm at 24 hours and 48 hours after transfection using microplate reader.
Wound Healing Assay
First of all, cells were plated into 6-well plates for 24 h and horizontal lines were evenly drawn at every 0.5-1cm on the back of the 6-wells plate. Secondly, the cells were exposed to transfection treatments with a serum-free culture medium was added and placed in a 37°C incubator. Finally, photos were taken at 0 h, 24 h with optical microscope and analyzed using ImageJ software (National Institutes of Health).
Transwell Assay
First, the transfected HCC cells were digested and collected, resuspended with 200ul serum free DMEM medium. And 20000 cells were seeded into 24-well upper Transwell chamber (8µm pore size) with Matrigel (BD, USA). Second, 600ul DMEM medium supplement with 20% FBS was added to the 24-well plate. Third, cells were cultured in a cell incubator for 24–72 hours. Finally, cells were washed, fixed, stained for taking photos under a microscope for analysis.
Flow Cytometry Analysis
Apoptosis was detected with flow cytometry. Cells were plated into 6-well plates for 24 h and exposed to transfection treatments for 48h. Cells were collected from the culture medium and resuspended with PBS. Then, cells were incubated using reagent from the Annexin V/PI apoptosis kit (BD, USA), according to the manufacturer’s protocol.
Statistical analysis
R v3.6.0 (https://www.r-project.org/) for clinical scales, analysis of gene expression. Statistical analysis was performed using SPSS 25.0 software (IBM Corp.) Results are expressed as the mean ± SD. The statistical differences among different groups were analyzed using a one-way ANOVA with Bonferroni post-hoc test. t-test was used for comparison between two groups. Differences in proportions between groups were examined by chi-square test. Spearman or Pearson test was used to determine correlations. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Data quantification was performed using Image J software and Image Lab software, and figures were drawn by GraphPad Prism 8.0 (GraphPad Software, Inc.).