Thirty paired HCC cancerous tissues and matched para-cancerous tissues were collected from patients of the First Affiliated Hospital, Zhejiang University School of Medicine, who underwent surgery between September 2016 and March 2017. The collected samples were all from patients who had histologically diagnosed as primary hepatocellular carcinoma and had not received prior treatments, such as TACE or chemotherapy before collection. The exclusion criteria were as follows: patients with other cancers, metastatic liver cancer (secondary liver cancer), previous liver transplantation, chemotherapy or TACE treatment. The research was permitted by the Medical Ethics Committee of the hospital. All tissues were immediately frozen at -80 °C. All patients’ information is listed in Table S1.
Cell lines and cell culture
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 with phosphate buffered saline. In addition, all cells were incubated in 5% CO2 at 37 °C. The Huh7-Luc cell line was constructed in our lab in a previous study.
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 the system changed according to cell status, and cell growth was measured by using a parameter termed the cell index (CI). Fifty microliters of complete media were added to the plates, and the baseline was measured. Then, 5×103 cells were seeded into every well and grown for approximately 80 h at 37 °C in 200 µL of culture media. The impedance was detected every 15 min for 72 h after seeding. Data were analyzed by xCELLigence software, and the cell index was normalized to the data recorded at the time of cell treatment.
RNA from cells and tissues was extracted by TRIzol (Takara #9109) according to the manufacturer’s protocol. Then, reverse transcription (RT) was performed using the 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 purchased from RiboBio Co., Ltd., China. The primer sequences used are 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 of 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. A total of 1×105 cells per well in 200 µL of serum-free culture medium was seeded into chambers after 48 h of transfection, and 600 µL of 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.
Cell migration ability was assessed with wound-healing assays. Hep3B cells (3× 105) and Huh7 cells (4 × 105) after 48 h of treatment 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, and 48 h. Three representative visual fields were selected from each dish, and the cell migration distances were measured with a microscope.
Colony formation assay
Five hundred cells after transfection were seeded into 6 cm petri dishes with complete medium, 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. After washing with deionized distilled water, the dishes were air dried at room temperature. Colonies were counted and photographed with a Molecular Imager® Gel DocTM XR+ imaging system (BIO-RAD). All assays were repeated three times.
The relationship of EIF4G2 and HCC prognosis was obtained from the Human Protein Atlas database. The EIF4G2 mRNA level data were obtained from the GEPIA database. miRNAs that can modulate EIF4G2 were predicted by the TargetScan, miRCODE, and starBase websites.
MiR-144 overexpression and ctrl lentivirus were purchased from GeneChem. Cells were seeded into 6-well plates at approximately 40-50% confluence, and 10 µL of lentivirus was added per well. Two micrograms/mL puromycin (MCE #NSC3056) was added to select infected cells after 48 h of transduction. Subsequently, cells were cultured under 1µg/mL puromycin to maintain stable infection.
SiRNA and vector infection
Si-RNAs of EIF4G2 and a negative control RNA were purchased from GenePharma. Cells were transfected with 50 nM siRNA 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 by GeneChem. Transfection of each vector was carried out using Lipofectamine®3000 (Invitrogen #L3000-015). U0126 (MCE #HY-12031) was purchased from MCE. In addition, 20 µM 0126 was used to inhibit the ERK pathway. Total RNA and protein were extracted after 48 h of treatment. The transfection efficiency was verified by RT-qPCR and Western blot. Sequences of the siRNAs are listed in table S3.
The wild-type (WT) and mutant (MUT) luciferase reporter vectors of the EIF4G2 3’-UTR were obtained from GeneChem. A total of 1x104 293T cells were seeded into 96-well plates, and then the WT and MUT reporter vectors and miR-144 overexpression vector were cotransfected with Lipofectamine 3000 into 293T cells. Forty-eight hours later, the firefly and Renilla luciferase activities were assessed by a GloMax microplate luminometer (Promega, USA) using a Dual-Glo® Luciferase Assay System kit (E2920, Promega, USA). Renilla luciferase activities were used to evaluate transfection efficiency.
Western Blotting Analysis
Protein was extracted from cells and tissues with RIPA lysis buffer (Applygen #C1053), and the protein concentration was measured with a BCA assay (Thermo Fisher, #23227). Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GenScript #M00657), transferred from the gel to 0.2 µm 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 approximately 14 h and incubated with secondary antibodies for 1 h at room temperature. The specific primary antibodies used in this paper were as follows: EIF4G2 (1:1000, CST #2182), p-ERK1/2 (1:1000, CST #4370), ERK1/2 (1:1000, CST #4695), p-p38 (1:1000, CST #4511), p38 (1:1000, CST #8690), p-JNK (1:1000, CST #9255), JNK (1:1000, CST #9252), and GAPDH (1:1000, CST #5174).
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 freely. Huh7-Luc cells (1 × 106) that had been transfected with si-EIF4G2, si-NC, miR-144 overexpression or ctrl lentivirus were injected subcutaneously into the backs of mice near their hind legs (7 mice pergroup). The left area was the ctrl treatment, and the right area was the 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 approximately 20 days by cervical dislocation. 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, Zhejiang University School of Medicine.
Tissue microarrays and Immunohistochemistry
The HCC tissue microarray chip, including total 89 pairs of HCC tumor tissues and matched para-carcinoma tissues, was purchased 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 was 100%. If a score was less than or equal to 1, we categorized it as the EIF4G2 low expression group, and if a score was greater than 1, we categorized it as the EIF4G2 high expression group.
RNA profiling and RNA-seq analysis
Total RNA was isolated and purified by TRIzol reagent following the protocal, and then 2×150bp paired-end sequencing (PE150) was performed on an Illumina Novaseq™ 6000. The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and p value < 0.05, and then GO enrichment and KEGG enrichment analyses were performed on the differentially expressed mRNAs.
Statistical analyses were performed with SPSS 18.0 and GraphPad Prism 8 software. Data were presented as the mean ± standard deviation (SD). In the HCC-TMA chip, chi-square tests were used to analyze EIF4G2 expression in HCC and matched tissues. Overall survival (OS) and disease-free survival (DFS) were assessed 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 a paired t test. HRs and 95% confidence intervals (CIs) for clinical correlative factors were investigated using the Cox regression models in univariate and multivariate analyses. A value of p<0.05 was considered to be statistically significant.