HCC patients
Thirty paired HCC cancerous tissues and matched para-cancer tissues were collected from the first affiliated hospital of Zhejiang university, who underwent surgery between September 2016 and March 2017. Patients in this study had not been accepted TACE or chemotherapy or any other treatments. The research was allowed by the Medical Ethics Committee of the hospital. All tissues were frozen at -80 °C immediately. All patients’ information was list in Table S1.
Cell lines and cell culture
The human HCC cell lines (Huh7, Huh7-Luc(Huh7 cells stably expressing luciferase), Hep3B) and 293T cells were cultured with Dulbecco's modified Eagle's medium (DMEM; HyCloneTM #AE29431636) and 10% fetal bovine serum (FBS; CORNING #35081006). The cell lines were harvested via trypsinization and washed by phosphate buffered saline. And all cells were incubated in 5% CO2 at 37 °C. Huh7-Luc cell line was constructed in our lab previous study[16].
Real-Time Cell Analysis (RTCA)
Cell proliferation assays were monitored by the xCELLigence Real-Time Cell Analysis S16 instrument and E-Plate 16 (ACEA Biosciences, Inc.) according to the manufacturers’ instructions. The electronic impedance of system changed according to cell status, then cell growth was measured by using a parameter termed the cell index (CI). 50 uL complete media was added into the plates and baseline was measured. Then 5×103 cells were seeded into every well and grown for about 80 h at 37 °C in 200 uL culture media. The impedance was detected every 15 min during 72 h after seeding. Data were analyzed by xCELLigence software and the cell index normalized to the data recorded at the time of cell treatment.
RT-qPCR
RNA of cells and tissues were extracted by Trizol (Takara #9109) according to the manufactures’ protocol. Then reverse transcription (RT) reaction was performed using PrimeScriptTM RT reagent kit with gDNA eraser (Takara #RR047A) according to the protocol. Quantitative real-time polymerase chain reaction (RT-qPCR) was conducted using TB GreenTM Premix Ex TaqTM (Tli RNaseH Plus), (Takara #RR420A). The results were quantified using the 2−ΔΔCt method. In the present study, GAPDH and U6 were used as reference genes to normalize EIF4G2 and miR-144-3p, respectively. The primers of miR-144-3p and U6 were bought from RiboBio Co., Ltd., China. The primer sequences used were listed in table S2.
Matrigel Invasion Assay
Cell invasion ability was assayed by 8mm Corning transwell insert chambers (Corning), which were coated with 50 μL Matrigel® (Corning®) and placed into 24-well plates. Then the 24-well plates were placed for 30 min at 4 °C, and incubated for 1-2 h at 37 °C. 1×105 cells per well in 200 uL serum-free culture medium were seeded into chambers after 48 h of transfection, and 600 uL DMEM with 10% FBS was inserted into the lower chamber. After 48 h, invaded cells were fixed with 4% paraformaldehyde (Servicebio #1101) for 30 min and stained with 0.1% crystal violet (Dawen Biotec) for 30 min. After removing cells on the upper surface, the invaded cells were imaged and counted.
Migration Assay
Cell migration ability was performed with wound-healing assays. Hep3B cells (3× 105) and Huh7 cells (4 × 105) after 48 h of transfection were seeded into 24 well plates. After cells reached complete confluence, linear wounds were made with 200 μL plastic pipette tips. And cells were cultured in DMEM with 1% FBS. Images were captured at 0, 12, 24, 48 h. Three representative visual fields were selected from each dish, and the cells migration distances were measured with microscope.
Colony formation assay
Five hundred cells after transfection were seeded into 6 cm petri dishes with complete media, and grown up to until visible colonies formed about 2 weeks. Cells were fixed with 4% paraformaldehyde for 30 min and then stained with Coomassie Brilliant Blue (FUDE Biological #FD0022) for 30 min. Washing with deionized distilled water, the dishes were air dried at room temperature. Colonies were counted and photographed with Molecular Imager® Gel DocTM XR+ imaging system (BIO-RAD). All assays were repeated three times.
Bioinformatics analysis
The relationship of EIF4G2 and HCC prognosis was from the Human Protein Atlas database. And the data of mRNA level of EIF4G2 was from GEPIA database. miRNAs which can modulate EIF4G2 were predicted by the website of TargetScan, miRCODE, and starBase.
Lentivirus transduction
MiR-144 overexpression and ctrl lentivirus were purchased from GeneChem. Cells were seeded into 6-well plates about 40-50% confluence, and adding 10 uL lentivirus per well. 2 ug/mL puromycin (MCE #NSC3056) were added to select infected cells after 48 h transduction. Subsequently, cells were cultured under 1 ug/mL puromycin to keep stably infected.
Si-RNA and vector transfection
Si-RNAs of EIF4G2 and a negative control RNA were purchased from Genepharma. Cells were transfected with 50 nM si-RNA for 48 h at 37 °C using the Lipofectamine® RNAiMAX (Invitrogen #13778-015) based on the manufacturer’s protocol. Overexpression vectors of EIF4G2 and miR-144 and corresponding ctrl vectors were designed and synthetized from GeneChem. Transfection of each vector was carried out using Lipofectamine®3000 (Invitrogen #L3000-015). Total RNA and protein were extracted after 48 h transfection. And transfection efficiency was verified by RT-qPCR and Western blot. Sequences of the si-RNAs were listed in table S3.
Luciferase assay
The wild-type (WT) and mutant (MUT) luciferase reporter vectors of EIF4G2 3’-UTR were obtained from GeneChem. 1x104 293T cells were seeded into 96-well plates, then the WT and MUT reporter vectors and miR-144 overexpression vector were co-transfected with Lipofectamine 3000 into 293T cells. 48 h later, the firefly and Renilla luciferase activities were assessed by GloMax microplate luminometer (Promega, USA) using Dual-Glo® Luciferase Assay System kit (E2920, Promega, USA). Renilla luciferase activities were used to evaluate transfection efficiency
Western Blotting Analysis
Protein of cells and tissues was extracted with RIPA lysis Buffer (Applygen #C1053), and protein concentration was measured with a BCA assay (Thermo Fisher, #23227). Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GenScript #M00657), then transferred from gel to 0.2 um polyvinyl difluoride membrane and blocked in TBS-T containing 5% bovine serum albumin (Haoke HK5021) or skim milk powder for 2 h. Then, the membrane was incubated with specific primary antibodies at 4 °C for about 14 h and incubated with secondary antibodies for 1 h at room temperature. The specific primary antibodies used in this paper are as follows: EIF4G2 (1:1000, CST #2182), p-ERK1/2 (1:1000, CST #4370), ERK1/2 (1:1000, CST #4695), GAPDH (1:1000, CST #5174).
Animal studies
BALB/cJGpt-Foxn1nu/Gpt (BALB/c) female mice (4 weeks old, 12-14 g) were purchased from GemPharmatech Co. Ltd (Nanjing, China), and housed in temperature-controlled and pathogen-free rooms. Water and food were provided free. Huh7-Luc cells (1 × 106) that had been transfected with si-EIF4G2, si-NC, miR-144 overexpression and ctrl lentivirus, were injected subcutaneously into back of mice near their hind legs (7 mice each group). And the left area was ctrl treatment, the right area was experimental treatment. Tumor size was measured every 2 days and tumor volume was calculated using this formula: volume= (length × width2)/2. Mice were sacrificed after about 20 days by cervical dislocation. And the tumors were removed and weighed and photographed. All protocols of animal studies were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhejiang University.
Tissue microarrays and Immunohistochemistry
The HCC tissue microarray chip including total 89 pairs of HCC tumor tissues and matched para-carcinoma tissues were bought from Shanghai Biochip Company Ltd. IHC staining was performed to assess EIF4G2 expression in HCC tissues according to the manufacturer’s protocol using anti-EIF4G2 antibody (dilution 1:400, abcam #ab97302). Each HCC-TMA score was analyzed based on the product of staining intensity and the percentage of positively stained cells. In this study, the positive rate is 100%. If a score less than or equal to 1, we categorized it as EIF4G2 low expression group, and if a score greater than 1, we categorized it as EIF4G2 high expression group.
Statistical analysis
Statistical analyses were performed with SPSS 18.0 and GraphPad Prism 8 software. Data were presented as the mean ± standard deviation (SD). In HCC-TMA chip, Chi-square tests were used to analyze the EIF4G2 expression in HCC and matched tissues. The overall survival (OS) and disease-free survival (DFS) were performed using the Kaplan–Meier method with the log-rank test. The correlation between the expression of EIF4G2 and PDL1 was analyzed by the Spearman’s rank correlation test. The correlation between the expression of miR-144 and EIF4G2 protein was analyzed by the Pearson’s test. The expression of miR-144 and EIF4G2 in 30 paired HCC tissues was performed using paired t test. HRs and 95% confidence intervals (CIs) for clinical correlative factors were investigated using the Cox regression models in univariate and multivariate analysis. Value of P<0.05 was considered to be statistically significant.